TRAF6, a well-known adapter molecule, plays pivotal role in TLR/IL-1R associated signaling pathway. Although TRAF6 has been shown to have oncogenic activity in various malignant tumors, the details remain unclear. In this study, we demonstrated that TRAF6 facilitates Ras (G12V) and EGF-induced cellular transformation through EGFR. Silencing of TRAF6 expression significantly downregulated AP-1 activity, as well as MMP-2,9 expression after EGF stimulation. Furthermore, we found that TRAF6 plays an essential role in cutaneous squamous cell carcinoma (cSCC) malignant phenotypes, affecting cell growth and migration. CD147/Basigin, a transmembrane glycoprotein belonging to the immunoglobulin superfamily, is over-expressed in tumors and induces tumorigenesis. Our results showed that CD147 formed complex with EGFR and TRAF6. Knockdown of TRAF6 disrupted the CD147-EGFR complex, thereby inducing EGFR endocytosis. Therefore, TRAF6 might be a novel molecular target for cSCC prevention or therapy. Although the incidence of cSCC is much lower than that of BCC in the general population, the clinical characteristics of cSCC manifest more aggressively. cSCC refers to the cellular malignant transformation and abnormal growth of keratinocyte cells, which are the major cell type in the epidermis. Unlike BCC, cSCC also shows aggressive behavior, which showed the risk for metastasis relevent to advanced high-risk lesions with, and ~4.0–12.5% of patients have nodal metastasis3. In addition, the increased incidence of recurrence and metastasis after surgical resection is associated with several features, including tumor diameters of >2 cm, deep invasion (>2 mm), localization to chronically damaged or diseased skin and poor histological differentiation4.Cutaneous squamous cell carcinoma (cSCC) is second incidence to basal cell carcinoma (BCC) among malignant non-melanoma skin cancersMost cSCC occurs on the head, neck, and extremities, where there is a large possibility of exposure to the sun, as ultraviolet (UV) exposure is the major cause and a direct contributor to the occurrence of cSCC. cSCC pathogenesis follows the classic tumor model, involving multiple steps from precancerous lesion, such as actinic keratosis (AK) to carcinoma in situ, final to invasive of cSCC. Subsequent steps include malignant transformation, abnormal cell growth, angiogenesis, invasion of the surrounding tissue and formation distant organ metastasis.5. After associating with its ligands, such as EGF or transforming growth factor-a (TGF-a), EGFR dimerizes and triggers auto-phosphorylation of tyrosine kinases, thus leading to activation of various intracellular downstream pathways, including the PI3K/AKT, RAF/MEK/ERK, and STAT3 signaling pathways6. Dysregulation of EGFR is closely linked to tumorigenesis and has been implicated in a number of tumors11. EGFR is over-expressed on cSCC cells, particularly in advanced or metastatic tumor tissue14. Genetic analysis has indicated that EGFR has a very low frequency of mutation in cSCC16, and RAS mutations are also very rarely observed in cSCC17. Mutated RAS may activate molecules downstream of EGFR, and consequently, inhibition of EGFR is largely ineffective in tumor entities with RAS mutations. Because of dysregulated EGFR activation in the absence of EGFR or RAS mutations, targeting EGFR is a promising therapeutic strategy in cSCC20.EGFR  is a transmembrane member of the ErbB receptor tyrosine kinase family, which is located on the cellular surface21. In addition, TRAF6 has an E3 ubiquitin ligase activity mediatesd conjugation of lysine-63 (K63)–linked polyubiquitin chains to proteins23. Recent studies have reported that TRAF6 promotes oncogenesis by inhibiting apoptosis and stimulating proliferation and invasion in cancer. TRAF6 alters the expression of Bcl2, Bax, and MMP9, thereby regulating cell apoptosis and invasive ability in gastric cancer24. TRAF6 upregulates HIF-1a expression and promote tumor angiogenesis in colon cancer25. Luo et al. have demonstrated that TRAF6 directly interacts with CD147, thereby promoting melanoma invasion and metastasis, whereas inhibition of TRAF6 expression or activity reverses the malignant phenotype of melanoma cells27. In addition, TRAF6 is highly expressed in human pancreatic cancer28, colon cancer29, gliomas30, breast cancer31, and lung cancer32. However, the role of TRAF6 in cSCC remains unknown. In this study, we found that TRAF6 is required for EGF-induced cell transformation and plays critical roles in cSCC cell growth and metastasis through EGFR signaling pathways.Tumor necrosis factor receptor-associated factor 6 (TRAF6), a member of the TRAF family, was first identified as an adaptor of the signals induced by the TNFR. The TRAF family comprises signal transducers of TLR/interleukin-1 (IL-1) family members, which triggers signaling transduction in innate immune responsesTRAF6 has a critical function in the LPS/IL-1β-induced signaling pathway through the TAK1-Ikkα/β pathway, but whether TRAF6 is involved in the oncogenic stimuli-induced transduction pathway remains unknown. Our findings showed that TRAF6 promotes Ras (G12V)-induced cell transformation in NIH3T3 cells. The NIH3T3 cells had been transfected with TRAF6 alone, Ras (G12V) alone or TRAF6 with Ras (G12V). As shown in Fig. Cell transformation is a critical step in the process of cSCC, and our above data showed that TRAF6 affects Ras (G12V) and EGF-induced cell transformation, as well as cell migration in HaCaT cells, thus suggesting that TRAF6 might be involved in cSCC pathogenesis. To investigate the effect of TRAF6 on the cSCC malignant phenotype, we generated a stable TRAF6-knockdown A431 cell line Fig. . As showTo validate the effect of TRAF6 on cSCC growth, we constructed xenograft models to test the role of TRAF6 in cell proliferation in vivo. In agreement with the in vitro data, silencing TRAF6 expression significantly blocked cSCC cell growth, thus resulting in smaller tumor sizes and delayed tumor formation Fig. . Histopa35. Given TRAF6 regulation of EGFR, we next analyzed MMP-2,9 expression in TRAF6-silenced HaCaT cells after EGF treatment. As shown in Fig. Our previous results indicated that TRAF6 promotes Ras (G12V) and EGF-induced cell transformation, as well as a malignant phenotype in A431 cells, thus suggesting that TRAF6 might, to some extent, exert role in EGF pathway. To investigate the details of the role of TRAF6 in the EGFR pathway, we used an EGFR Signaling Antibody Array and found that the expression of total EGFR and p-EGFR was significantly decreased in TRAF6-knockdown HaCaT cells Fig. . To confAP-1 reporter gene with different amount of TRAF6-WT or TRAF6-DN (ring domain mutation), as shown in Fig. AP-1 had been dramatically raised with dose-dependent manner in cells expressing TRAF6-WT but not in those expressing TRAF6-DN. Next, to confirm the effect of TRAF6 on AP-1 activity, the AP-1 reporter gene together with Renilla luciferase gene were transfected into TRAF6 expression silence HaCaT cells and found that AP-1 activity was downregulated in TRAF6 low-expression HaCaT cells . Our previous results had demonstrated that TRAF6 directly interacts with CD147. CD147 is a cell surface protein belonging to the immunoglobulin super-family involved various tumor malignant phenotypeslex Fig. , thus inlex Fig. . We alsolex Fig. .Fig. 5TRUnder ligand stimulation, endocytosis of EGFR occurs and leads to a shift from diffuse membrane staining to punctate cytoplasmic staining (representing endocytic vesicles). To understand the role of TRAF6 and CD147 in EGFR trafficking, we utilized a fluorescence-conjugated primary antibody to track EGFR and examined the effect of TRAF6, CD147 on EGFR protein stability. We found that knockdown of TRAF6 and CD147 accelerated EGFR-protein degradation Fig.  and EGFR44. In the IGF-induced pathway, TRAF6 acts as a direct E3 ligase, ubiquitinating AKT, and thereby inducing AKT cellular membrane recruitment and phosphorylation leading to activation45.Epidermal cell transformation plays a critical stage in cSCC development that involves oncogenic key molecules. In this study, we found that TRAF6 plays a key role in cell transformation. Although over-expression of TRAF6 alone did not effect on foci formation in NIH3T3 cells, more foci were observed with a combination of TRAF6 and Ras (G12V), thus indicating that TRAF6 enhances the cell transformation ability driven by oncogenes Fig. . Slienci49. EGFR exerts these functions through multiple signaling cascades. After ligand binding, tyrosine residues at the intracellular domain of EGFR are auto-phosphorylated, thus creating docking sites for adapter proteins, such as Grb2 or GAB1 (4). For example, after binding to EGFR, Grb2 recruits SoS1 (a Ras guanine nucleotide exchange factor) cell membrane localization, thereby activating the Ras/Raf/ERK signaling cascade51. AP-1 is a downstream convergence point of different signaling cascades and is required for EGF-induced cell transformation and tumor development. In addition, EGFR has been reported to regulate invasion or migration by regulating MMP expression, in a manner dependent on AP-1 activity54. MMPs have a critical role in ECM remodeling, embryonic development and wound healing in physiological conditions and facilitate tumor cell migration and metastasis through ECM degradation, modulation of cell adhesion and promotion of angiogenesis. We found that AP-1 activity was decreased in both TRAF6-DN and TRAF6-knockdown cells  and inhibits CDKs activity, thus leading to growth arrest at G2 phase of the cell cycle61. CD147/BSG, a transmembrane glycoprotein belonging to the immunoglobulin superfamily, has various physiological functions, such as fertilization, spermatogenesis, T-cell development, formation of the blood brain barrier and retinal development62. CD147 expression had been found to be elevated in different tumors, such as liver, pancreatic, lung, and colon cancers65. Moreover, CD147 is overexpressed in both head and neck cancer and oral squamous cell carcinomas and is associated with head and neck cancer metastasis and patient survival69. Interestingly, blocking CD147 expression with anti-CD147 mAb or si-CD147 inhibits EGFR expression and leads to cSCC growth suppression in vitro and in vivo70. Targeting both EGFR and CD147 efficiently decreases HNSCC cell proliferation and migration71. Our results showed that CD147 directly associates with EGFR, as well as with TRAF6 in HaCaT and A431 cells - and EGF-induced cellular transformation through EGFR. Silencing TRAF6 expression significantly downregulates AP-1 activity, as well as MMP-2,9 expression after EGF stimulation. Furthermore, we confirmed that TRAF6 promotes a malignant cSCC phenotype, thus affecting cell growth and migration. In addition, CD147 associates with EGFR and TRAF6, and knockdown of TRAF6 disrupts the CD147-EGFR complex. Therefore, TRAF6 might be a novel molecular target for cSCC prevention or therapy.The cell culture reagents, such as medium  and fetal bovine serum (FBS) were obtained from Life Technologies, Inc. . The TRAF6 antibody  was used by 1:500, anti-GAPDH  was prepared at 1:3000; the antibody of c-Myc, CD147  had been prepared by 1:500 dilution; The Flag antibody  was diluted at 1:5000 and the no-EGFR, p-EGFR, no-STAT3, p-STAT3  were used at 1:1000 dilution. The EGFR Signaling Antibody Array Kit (#12622) was purchased from Cell Signaling Technology (Danvers) Co., Ltd.The NIH3T3, Immortalized human keratinocyte cells (HaCaT) and human epidermoid carcinoma cells (A431) were purchased from the American Type Culture Collection (ATCC) and maintained in DMEM medium . The cytogenetically testing and authentication was performed by STR-PCR. For transfection experiments, cells were co-transfected with different plasmids using TurboFect  following the manufacturer’s protocol.74. Briefly, pLKO1-TRAF6 plasmids together with pspAX2 and pMD2G were transfected into 293T cells, after 48 and 72 h transfection, the supernatant containing lentiviruses was collected, and then, along with 10 μg/ml polybrene, was perfomred to infect A431 and HaCaT cells. After infection 16 h, the infected cells were continued to culture in medium with 1.0 μg/ml puromycin until the control cells (uninfected) died .The stable knockdown TRAF6 expression cells in HaCaT or A431 were generated as described previousThe extracts from cells were lysed with NP40 buffer containing protease inhibitor cocktail. For immunoprecipitation, extracts were pretreatment with 20 μl of agarose A/G-Sepharose beads (Beyotime Institute of Biotechnology) and incubated for 1 h at 4 °C with gentle shaking. Supernatants were collected and removed to a new tube, and then, 1.5 μg of antibodies and 40 μl of agarose A/G-Sepharose beads were added to the supernatants, and the mixture was rotated overnight at 4 °C. The beads were then washed three times in NP40 buffer, and the proteins analyzed by western blotting.The cells were collected and lysed by modified RIPA buffer containing 50 mM Tris-Cl pH 8.0, 150 mM NaCl, 0.5% NP-40, and protease-inhibitor cocktail. The protein of concentration had been tested with a BCA Kit, and appropriate amounts of protein were prepared for SDS-PAGE and then transferred to a PVDF membrane (Millipore). The membranes were blocked for 1 h with 5% non-fat dry milk, and then incubated with primary antibodies for overnight at 4 °C. The membranes washed with phosphate-buffered saline (PBS) buffer with 0.1% Tween 20 (PBS-T), reacted with horseradish peroxidase-conjugated secondary antibodies for 1 h, and visualized using an enhanced chemiluminescence substrate. The HaCaT cells were grown to 90% confluence and treated with serum-free medium for overnight. After treated with EGF at 100 ng/ml, cell extracts were prepared and analyzed by PathScan EGFR Signaling Antibody Array Kit (#12622). The antibody array was performed according to the kit protocols recommended by the manufacturer and detected with an imaging system .75. In brief, the H-RasG12V (100 ng) and TRAF6 (2.0 μg) were transiently transfected into NIH3T3 cells at six wells. After transfection 36 h, cells were splited into 10 cm dishes and cultured in DMEM for 2 week. The medium had been changed every 3 d. The Foci were fixed and stained with 0.5% crystal violet. Cell colony was calculated with a microscope (×40) and each assay was performed in triplicate.The protocol of H-Ras(G12V) mediated cellular transformation followed previous describedAP-1-Luc and SV-40-Renilla-Luc . After 20 h transfection, cells were continued to culture with serum-free medium for 16 h and then treated with EGF (100 ng/ml) for indicated time points. Cell lysates were analyzed by firefly and Renilla luciferase activities with a dual-luciferase assay kit (Promega) following protocol.Cells were transfected with p21 primers used were as follows: (forward) 5′-ACTACCACTCACCCGCAGAC-3′ and (reverse) 5′-CCAGGTCCGTGCAGAAGT-3′.The RNA was prepared from TRAF6 knocking-down HaCaT cells by the Trizol reagent. A total of 3 µg RNA was used as a template for the reverse transcription reaction (Invitrogen). The 76. Cells mixed with Basal Medium Eagle (BME) medium containing 10% FBS and 0.33% agar, in presence of EGF or not, and then, seeded into 6-well plates (8 × 103/ml/well) with coated with 3 ml of solidified BME medium(10% FBS and 0.5% agar). Colonies were scored with a microscope and ImageJ software.The EGF-induced cell transformation assay was performed as mentioned previous3/well cells were cultured in 96-well plates in 100 µl DMEM; living cells were counted from 0 to 96 h by analyzed with 20 μl of sterile MTS dye each day. Colorimetric analysis was performed at 490 nm was measured using a microplate reader. Each cell line was established in five replicates, and each analysis was repeated three times.The 2 × 1077, and the animal protocol was approved by the Ethics Committee of Xiangya Hospital . The A431 cells with knockdown of TRAF6 expression by sh-Mock or sh-TRAF6#1&#2 were collected and washed by PBS, and then resuspended by serum-free medium, the 4 or 6-weeks-old male nude mice  had been subcutaneously injected with tumor cells (5 × 106/0.15 ml) on the right flanks. Tumor diameters and weights were recorded twice a week, and tumor volumes (mm3) were calculated by length × width × height × 0.5326. Tumor growth curves were drawn for each group (mean ± S.D.).Xenograft tumor models were established as mentioned previously4 cells in serum-free medium were put into the upper chamber, and then 500 µl of DMEM plus 20% FBS was added into the lower chambers. After a 16 h of incubation at the temperature of 37 °C, cells in the chamber were fixed with formaldehyde for 15 min and stained with 0.1% crystal violet. Cells passed through the transwell membrane were finally counted with a microscope (×40). Five fields were selected randomly at least. Each assay was performed in triplicate.To evaluate the ability of migration in HaCaT and A431 cell lines, an 8-mm-pore size chamber  was used. Briefly, a number of 5 × 105 cells/well, and a straight line were scratched on the cell monolayer was by a 200-μl pipette tip. Then cells were washed by PBS three times to remove debris. Finally, cells were cultured in complete medium and photographed at 24 h and 48 h.Cells were cultured with complete medium in a 6-well plate at a density of 1 × 10Collecting cells first, and washing with PBS, then fixing in ice-cold 70% ethanol in PBS and centrifuged at 4 °C overnight. Next, cells were centrifuged, resuspended with cold PBS and incubated in RNase at 37 °C for 30 min. Then cells were added with propidium iodide and incubated for 30 min at 37 °C. The cell cycle distribution was analyzed by flow cytometry (FCM).Tumor samples from nude mice were cut and mounted on glass slides. Antigen retrieval was operated at 95 °C for 5 min in the microwave oven and cooling to room temperature, deparaffinized sections were treated with 3% hydrogen peroxide for 10 min. The slices were then washed in PBS for three times, 5 min each, and blocked in goat serum for 1 h. This was followed by the addition of anti-Ki-67 or EGFR antibody  in a humidified chamber overnight at 4 °C. After being washed by PBS, the slices were applied with a biotin-conjugated secondary antibody for 20 min and then peroxidase-conjugated streptavidin for an additional 30 min. After that, 3,3′-diaminobenzidine tetrahydrochloride was used to visualize the reaction and then the slices were counterstained with hematoxylin. Immunohistochemistry was performed at least three times.Cells seeded in 18-mm cover glasses were washed with ice-cold 1× PBS. After EGF stimulation, 4% paraformaldehyde fixed cells for 15 min at room temperature. Cells were washed by 1× PBS for three times and then permeabilized with 0.5% Triton X-100 for 15 min at room temperature. Cells were blocked by 1% BSA for 15 min and incubated with anti-EGFR  in 1% BSA overnight at 4 °C. Afterwards, the cover glasses were washed by 1× PBS for three times. Cells were counterstained with DAPI (0.5 μg/ml) for 30 min at room temperature. After being washed, the cover glass was mounted through fluorescent mounting medium. Thereafter, fluorescence microscopy images of cells were captured using a Leica SP8 Confocal laser scanning microscope.t-test or one-way ANOVA to examine the statistical differences. The p-value <0.05 was believed to be statistically significant.The statistical results were represented with mean ± S.D. and analyzed by Student’s supplementary figure legendsFigure 1


In the current study, we examined the clinical, prognostic and molecular correlates of increased ALP and aspartate aminotransferase (AST) in PMF.Hepatomegaly is a common finding in primary myelofibrosis (PMF),3 Cytogenetic and mutational analyses were performed according to previously described methods.4 ALP and AST were chosen as markers of liver function abnormality, based on test availability in the study population. On the basis of preliminary survival analysis and also to be consistent with formal Common Terminology Criteria for Adverse Events (CTCAEs), the optimal cutoff value chosen for abnormally increased ALP was ⩾grade 2 elevation per CTCAE (287 U/l), which is above 2.5-fold increase from the upper limit of the normal range for our institution (45–115 U/l). The optimal cutoff value chosen for AST was ⩾grade 1 elevation per CTCAE . Targeted next-generation sequencing was used to screen for prognostically relevant mutations.5 Statistical analyses considered clinical and laboratory data collected at the time of diagnosis or referral to the Mayo Clinic. Differences in the distribution of continuous variables between categories were analyzed by Mann–Whitney test. Patient groups with nominal variables were compared by χ2-test. Cox proportional hazard regression model was used for multivariable analysis. P-values <0.05 were considered significant. The Stat View  statistical package was used for all calculations.Study patients were selected from our institutional database of myeloproliferative neoplasms and fulfilled the 2016 World Health Organization criteria for the diagnosis of PMF.6 risk distributions were 16% low, 17% intermediate-1, 39% intermediate-2 and 28% high. Driver mutation distributions were 64% JAK2, 17% CALR type 1/type 1-like, 4% CALR type 2/type 2-like, 5% MPL and 10% triple negative. Cytogenetic studies, available in 390 patients, were abnormal in 36%. Other mutations that were concomitantly analyzed included ASXL1 (n=269), SF3B1 (n=228), U2AF1 (n=257), SRSF2 (n=269) and TET2 (n=92); their respective frequencies were 39, 6, 16, 16 and 16%.A total of 398 patients with PMF  were considered and their clinical and laboratory characteristics are listed in 9/l; P=0.0004) and increased AST (P=0.04); no other associations were apparent. AST ⩾49 U/l was also associated with leukocytosis as a continuous variable (P=0.0003) and leukocyte count >25 × 109/l (P=0.003), as well as increased lactate dehydrogenase (P<0.0001), increased bilirubin (P=0.008), increased ALP (P=0.0035), increased alanine transaminase (P=0.001) and SRSF2 mutations .The study cohort was stratified according to the presence of increased ALP ⩾grade 2 , and categorical variables with the respective cutoff levels of 287 U/l and 49 U/l ; the significant difference in survival was independent of DIPSS-plus and of driver mutational status for ALP as continuous (P=0.004) and categorical variable , but not for AST (P=0.11). Increased serum ALP or AST did not affect leukemia-free survival .After a median follow-up of 3 years, 236 (59%) deaths and 36 (9%) leukemic transformations were documented. In univariate analysis, increased ALP  and AST χ, the upper normal limit. Furthermore, the lack of effect on leukemia-free survival suggests that the adverse prognosis associated with ⩾grade 2 increases in serum ALP might be related to excess deaths from disease comorbidity rather than clonal evolution, but additional studies are needed to validate our observations and provide additional insight in the subject matter.In the current study, we showed that increased level of serum ALP is relatively common in PMF occurring in almost half of the patients at the time of referral, whereas the incidence of elevated serum AST was much less frequent at 9%. The study also shows that the magnitude of the abnormalities were mostly grade 1 for AST, but ⩾grade 2 for ALP in ~7% of the patients. In addition, our study demonstrates significant associations with marked leukocytosis for both ALP and AST. Of note, no other associations were apparent for ALP, but the multiple other associations for AST, which included serum LDH, suggest an indirect effect from the associated leukocytosis. Finally, we demonstrate a DIPSS-plus and driver mutational status independent adverse effect on survival from ALP but not AST. We conclude, therefore, that elevated ALP is frequent in PMF and prognostically relevant when it exceeds 2.5


Off-line, we derived indexes of SVR, cardiac output (CO) and baroreflex sensitivity from the continuous BP recordings (Modelflow). During oxygen supplementation, systolic, and diastolic BP both increased dose-dependently from 128 ± 24 and 72 ± 19 mmHg respectively at baseline to 141 ± 23 (p < 0.001) and 80 ± 21 mmHg (p < 0.001) at 1.0 ATA oxygen. Comparing baseline and 1.0 ATA oxygen, SVR increased from 1440 ± 546 to 1745 ± 710 dyn·s/cm5 (p = 0.009), heart rate decreased from 60 ± 8 to 58 ± 6 bpm (p < 0.001) and CO from 5.0 ± 1.3 to 4.6 ± 1.1 L/min (p = 0.02). Baroreflex sensitivity remained unchanged (13 ± 13 to 15 ± 12 ms/mmHg). These blood pressure effects were absent in a negative control group of eight young healthy subjects. We conclude that oxygen supplementation in CKD patients causes a non-baroreflex mediated increased in SVR and blood pressure.Renal hypoxia is thought to be an important pathophysiological factor in the progression of chronic kidney disease (CKD) and the associated hypertension. In a previous study among CKD patients, supplementation with 100% oxygen reduced sympathetic nerve activity (SNA) and lowered blood pressure (BP). We aimed to assess the underlying haemodynamic modulation and hypothesized a decreased systemic vascular resistance (SVR). To that end, 19 CKD patients were studied during 15-min intervals of increasing partial oxygen pressure (ppO Hypertension is a hallmark of chronic kidney disease (CKD). There is substantial evidence that this can be attributed to increased sympathetic nerve activity (SNA) . Of all patients, values of hemoglobin and proteinuria were available from clinical routine testing within 3 months before the study. Baseline characteristics, including medication use and disease background are given in Table 2). The study was carried out in accordance with the Declaration of Helsinki of the World Medical Association (2013). The Medical Ethics Review Committee of the Academic Medical Center  approved the study protocol. Before inclusion all participants provided written informed consent.We studied 19 CKD patients  0.21 ATA, was provided over the mask at 15 L/min for another 15 min. Thereafter the ppO2 in the breathing gas was increased to 50% O2 (ppO2 0.5 ATA) and 100% O2 (ppO2 1.0 ATA) respectively again for 15 min at each dose , a non-rebreathing mask was positioned over nose and mouth. Blood pressure measurement (see below) was continued while room air, partial pressure of oxygen (ppO2 0.21 ATA), under hyperbaric conditions at 2.4 ATA (ppO2 0.5 ATA) and at 2.4 ATA during 100% oxygen supplementation  at atmospheric pressure ppO 0.21 ATADuring all sessions, continuous blood pressure was measured using finger arterial photo-plethysmography . The device has been validated for use in CKD patients  was systolic BP (SBP) minus diastolic BP (DBP). All hemodynamic parameters were derived from the last minute of the measurements at baseline and at each oxygen dose. Time domain cross-correlation baroreflex sensitivity (xBRS) was calculated from the same intervals as the other parameters, using dedicated software . This algorithm provides a validated beat-to-beat estimate of left ventricular stroke volume (SV), based on a nonlinear 3-element model of the input impedance of the aorta  = 12.6, p < 0.001 for SBP and F = 8.8, p < 0.001 for DBP  = 5.8, p = 0.002, Figure 2, F = 25.1, p < 0.001] and CO  decreased during oxygen supplementation  = 4.3, p = 0.009, Figure 2 , DBP , MAP  and PP , did not change  = 13.0; p < 0.001] and CO  decreased with oxygen supplementation  = 0.884; p = 0.47, Figure During the normobaric oxygen challenge in the control group, SVR increased significantly from 903 at baseline to 985 dyn·s/cm1 Figure . SBP [F Oxygen supplementation causes a dose-dependent blood pressure increase among CKD patients. (2) This blood pressure increase is caused by an SVR increase. (3) The simultaneous HR decrease with unchanged baroreflex sensitivity indicates that the SVR increase is caused by a direct vascular effect of the increased plasma ppOOur results seem to contradict previous findings in CKD patients  its ability to simultaneously hinder vasodilatation by reducing nitric oxide (NO) bioavailability  do not support nor exclude the existence of a CKD-kidney specific hypoxic triggering of  chemo receptors. The overwhelming effects of oxygen on systemic vasoconstriction render the experimental set-up unsuitable to detect any possible subtle effects of kidney specific oxygenation on sympathetic outflow.A possible clinical implication of these results is that oxygen supplementation might act as a cardiovascular stressor in CKD patients. Interestingly, this is in line with observations that oxygen supplementation in selected clinical patients is associated with worse outcome Kones, . Additio2 partial pressure during oxygen supplementation. However, this has previously been shown not to be influenced by oxygen supplementation  pronounced effect on RAAS activity or intrarenal oxygen delivery. However, this may only have blunted the hemodynamic effects and thereby would not have affected our eventual conclusions, especially since patients acted as their own control. The same holds for the heterogeneous distributed baseline parameters  in our relatively small patient group. Secondly, we did not assess changes in COWe have shown that oxygen supplementation in CKD patients increases blood pressure in a dose dependent fashion. This response is mediated by an increase in SVR, likely as the result of hyperoxic vasoconstriction independent of baroreflex function.Study conception and design by RB, RH, ES, CK; Data acquisition, analysis and/or interpretation by RB, MÇ, RH, JL, CK; Drafting of the manuscript by RB, CK; Revising by MÇ, RH, JL, ES, CK; Final approval of manuscript provided by RB, MÇ, RH, JL, ES, CK.This project was funded by the Dutch Kidney Foundation (Project KJPB 12.029 to CP). CP is supported by the Netherlands Organization for Health Research and Development .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Whitt et al. identify the Ca2+ channel subtypes that activate BK current in the suprachiasmatic nucleus to enable time of day to be encoded by the circadian clock.Large conductance K + (BK) channels are expressed widely in neurons, where their activation is regulated by membrane depolarization and intracellular Ca2+ (Ca2+i). To enable this regulation, BK channels functionally couple to both voltage-gated Ca2+ channels (VGCCs) and channels mediating Ca2+ release from intracellular stores. However, the relationship between BK channels and their specific Ca2+ source for particular patterns of excitability is not well understood. In neurons within the suprachiasmatic nucleus (SCN)—the brain’s circadian clock—BK current, VGCC current, and Ca2+i are diurnally regulated, but paradoxically, BK current is greatest at night when VGCC current and Ca2+i are reduced. Here, to determine whether diurnal regulation of Ca2+ is relevant for BK channel activation, we combine pharmacology with day and night patch-clamp recordings in acute slices of SCN. We find that activation of BK current depends primarily on three types of channels but that the relative contribution changes between day and night. BK current can be abrogated with nimodipine during the day but not at night, establishing that L-type Ca2+ channels (LTCCs) are the primary daytime Ca2+ source for BK activation. In contrast, dantrolene causes a significant decrease in BK current at night, suggesting that nighttime BK activation is driven by ryanodine receptor (RyR)–mediated Ca2+i release. The N- and P/Q-type Ca2+ channel blocker ω-conotoxin MVIIC causes a smaller reduction of BK current that does not differ between day and night. Finally, inhibition of LTCCs, but not RyRs, eliminates BK inactivation, but the BK β2 subunit was not required for activation of BK current by LTCCs. These data reveal a dynamic coupling strategy between BK channels and their Ca2+ sources in the SCN, contributing to diurnal regulation of SCN excitability.Large conductance K BK channels are activated via membrane depolarization and increases in local intracellular Ca2+  or channels mediating Ca2+ release from intracellular stores in local nanodomains  receptors  are inhibited during the day  of the hypothalamus, the brain’s internal “clock,” BK currents are present alongside all the major VGCC and intracellular Ca the day . In cont the day , indicat neurons ;. L-type the day , suggest the day . Cytosolr stores , is also results , 2016. N results . Despite2+ sources are responsible for BK current activation in SCN neurons and whether there is evidence that circadian regulation of BK-Ca2+ coupling contributes to the day-versus-night difference in BK current properties and action potential firing  mice  at 25°C. SCN neurons were recorded in whole-cell configuration as described . Rs was compensated at 80%.Acute SCN slices were prepared as described , at Zeitescribed  from neu+ currents were recorded in 1 µM tetrodotoxin  ± focally perfused Ca2+ channel modulators: 10 µM nimodipine , 10 µM dantrolene , 5 µM thapsigargin , 3 µM ω-conotoxin MVIIC , 5 µM Bay K 8644 , or 100 nM Ryan . Stocks were prepared at 1,000× in DMSO  or water . Currents were elicited from a holding potential of −90 mV, stepping from −110 to 90 mV for 150 ms in 20-mV increments. BK currents were isolated by current subtraction after focal application of the BK antagonist 10 µM Pax (Alomone). Three currents were averaged per cell and normalized to cell capacitance, which did not differ between day and night or between WT and β2 KO (i) and sustained (BKs) currents were categorized by using the current ratio (peak to 30 ms) from 90-mV step , corresponding to τinact values <110 ms. V1/2 values were determined from Boltzmann fits of the I/Imax relationships .In voltage-clamp mode, total voltage-activated Knd β2 KO . Voltage2+ current recordings in whole-cell voltage-clamp mode, the internal solution was (in mM) 115 cesium gluconate, 10 tetraethylammonium chloride, 10 HEPES buffer, 0.5 EGTA, 2 MgCl2, 20 sodium phosphocreatine, 2 Na2ATP, and 0.3 Na3 GTP, pH 7.3, and bath (mM) 68 NaCl, 3.5 KCl, 1 NaH2PO4, 26.2 NaHCO3, 1.3 MgSO4, 2.5 CaCl2, 10 D(+)-glucose, 60 tetraethylammonium chloride, and 3 CsCl (pH 7.4) and 1 µM TTX. Rs was compensated at 60%. Inward Ca2+ currents were elicited by using the same voltage protocol as for BK currents (in 10-mV increments) at baseline and after 10 µM Nim or 3 µM MVIIC. Three currents were averaged per cell and normalized to cell capacitance.For Ca+ currents, and the data were acquired in 10-s sweeps. Frequency was calculated as the mean of each sweep. Input resistance and resting membrane potential were recorded in 1 µM TTX and calculated as previously described , and Fisher’s exact test categorical data .Group means are reported ± SEM. Reported numbers in figure legends are the number of neurons recorded, with 1–6 neurons per animal. Data for each condition was derived from a minimum of two animals. Statistical significance was determined at P < 0.05 by using Origin (OriginLab) or SPSS v19 (IBM) for unpaired Student’s 2+ channel inhibitors. Fig. S2 contains a comparison of the relative inhibition of BK current from WT and β2 KO SCN neurons during the day and night in Nim and Dan. Fig. S3 is a schematic model depicting a hypothesis for functional coupling of BK and Ca2+ channels between day and night SCN neurons. Fig. S1 shows data for passive membrane properties of day and night SCN neurons with Can = 65; night: 0.51 ± 0.05 Hz, n = 65; P < 0.05, t test), resting membrane potential , and input resistance , when firing was abolished by the application of 1 µM TTX. Inhibition of BK channel activity has been shown to regulate each of these parameters, with more significant effects at night compared with during the day  and cytosolic Ca2+i levels have been reported to be higher during the day than during the night , although the difference was not statistically significant under these conditions.First, because the Cais study . Macrosc studies , we founn = 13; night: −10 ± 2 pA/pF, n = 9; P < 0.05, t test). In contrast, the N- and P/Q-type Ca2+ current, isolated with MVIIC, was not different between day and night . These results show the activation of VGCC current under conditions used to record BK current is, at least in part, paradoxically inversely phased. Because VGCC and LTCC currents decrease at night without a concomitant decrease in BK current, it suggests that BK activation may be influenced by other mechanisms  and intracellular RyRs (Dan), local Ca2+ sources that are known to be coupled to BK channel activation in other cell types . During the day, BK currents recorded in Nim were reduced by 77%  and reduced the V1/2 values  SCNs  suggests that, although the β2 subunit is not globally required for activation by LTCCs during the day, it may modify coupling by either influencing the physical association between BK and LTCC or RyR channels or the functional response of BK channels to intracellular Ca2+ influx.The finding that increasing LTCC activity can alter BK inactivation also raises the possibility that the β2 subunit, which confers inactivation of SCN BK currents , plays as in SCN . To test/−) SCNs . During /−) SCNs . The sen/−) SCNs , the rel/−) SCNs . Nim dec/−) SCNs , which w/−) SCNs . Similar/−) SCNs , compare/−) SCNs . Concurr/−) SCNs , from 22/−) SCNs . Notably/−) SCNs  as it di/−) SCNs . The cha2+ . Thus, despite the mixed consequences of LTCC activation that may converge on action potential activity, at least half of Nim’s effect on daytime firing can be attributed to BK channels. Assuming each of these cells had a BKs current in the presence of Nim .The results rule out a model where circadian regulation of a single Cand night . These vchannels . Instead2+ source, which lose all BK current when that source is inhibited, and those where BK channels are activated by multiple sources, which display reduced BK current in the presence of individual inhibitors. This model based on multifarious coupling could account for some of the heterogeneity observed in this study in the peak BK current values between neurons recorded at the same time point and may also factor into the difference in the sensitivity of the BK current to the nonselective VGCC blocker cadmium observed in a prior study . However, the molecular mechanisms behind the nighttime reductions in VGCC and LTCC currents have not yet been established .Although overall the individual roles and interaction of these two Caer cells . Still, channels . The pren levels . Moreoven levels , providi2+ in SCN intracellular signaling is currently an open question. Ca2+i is subject to circadian regulation in SCN neurons (2+i rhythms (2+ signaling. BK channels are positioned to bridge this gap by virtue of their Ca2+-dependent activation and central role in the circadian oscillation in SCN firing (The role of Ca neurons , but its neurons , 2017a.  rhythms , suggestN firing .2+ influx through either of these sources could alter cellular excitability through multiple mechanisms. Our results suggest these mixed effects could be partially accounted for by considering whether the cells coexpress BK channels and whether those channels inactivate. For example, although part of the decrease in firing with Nim was recoverable by inhibiting BK channels, LTCCs likely also contribute to higher daytime firing by directly regulating membrane depolarization (2+ sources contribute to action potential firing rate through BK channels will require future studies.The results here identify that LTCCs, and the more novel finding that neuronal RyRs, may both contribute to SCN excitability through activation of BK channels. Supporting this hypothesis, the direction and time windows of Dan’s effect on firing matches the basic predictions for being mediated by BK channels . Howeverrization . Becauserization , sorting2+-dependent activation pathways converging on BK are strategically positioned to preserve channel activity over the circadian cycle.Taken altogether, despite the changes in the firing rate with LTCC and RyR inhibition reported here and in previous studies, independently, neither LTCC nor RyR inhibition fully eliminates the day-versus-night difference in firing or SCN rhythmicity  like the


Tctn3 belongs to the Tectonic (Tctn) family and is a single-pass membrane protein localized at the transition zone of primary cilia as an important component of ciliopathy-related protein complexes. Previous studies showed that mutations in Tctn1 and Tctn2, two members of the tectonic family, have been reported to disrupt neural tube development in humans and mice, but the functions of Tctn3 in brain development remain elusive. In this study, Tctn3 knockout (KO) mice were generated by utilizing the piggyBac (PB) transposon system. We found that Tctn3 KO mice exhibited abnormal global development, including prenatal lethality, microphthalmia, polysyndactyly, and abnormal head, sternum, and neural tube, whereas Tctn3 heterozygous KO mice did not show abnormal development or behaviors. Further, we found that the mRNA levels of Gli1 and Ptch1, downstream signaling components of the Shh pathway, were significantly reduced. Likewise, neural tube patterning-related proteins, such as Shh, Foxa2, and Nkx2.2, were altered in their distribution. Interestingly, Tctn3 KO led to significant changes in apoptosis-related proteins, including Bcl-2, Bax, and cleaved PARP1, resulting in reduced numbers of neuronal cells in embryonic brains. Tctn3 KO inhibited the PI3K/Akt signaling pathway but not the mTOR-dependent pathway. The small molecule SC79, a specific Akt activator, blocked apoptotic cell death in primary mouse embryonic fibroblasts from Tctn3 KO mice. Finally, NPHP1, a protein with anti-apoptotic ability, was found to form a complex with Tctn3, and its levels were decreased in Tctn3 KO mice. In conclusion, our results show that Tctn3 KO disrupts the Shh signaling pathway and neural tube patterning, resulting in abnormal embryonic development, cellular apoptosis, and prenatal death in mice. Among causative genes for these diseases, tectonic mutations have been shown to be important in facilitating ciliopathies7. Tectonic (Tctn) family proteins are a group of proteins existing in the cilium transition zone (TZ), including Tctn1, Tctn2, and Tctn39. Tctn proteins are considered to play a vital role in trafficking proteins into the cilia and are required for the development of cilia and ciliogenesis11, although how Tctn proteins facilitate selective transportation of ciliary proteins to cilia remains unknown. Previous studies have shown that Tctn1 and Tctn2 mutations result in abnormal development in mice, such as defects in eye development, neural tube, and limbs12. Owing to the high sequence homology of the three Tctn members, Tctn3 mutations may share similar phenotypes if the functions of Tctn3 are not redundant with those of Tctn1 and Tctn2. So far, the functions of Tctn3 remain unclear, requiring further investigation.Loss of a single protein component within a ciliary protein complex disrupts the integrity of that complex and causes defects in the membrane protein composition of the cilia12, which is essential for cell fate and organ development17. Specifically, both in tectonics mutated primary mouse embryonic fibroblasts (pMEFs) and mice, Ptch and Gli1, widely used transcriptional targets of the SHH pathway, decline significantly12. Studies have shown that Tctn1 is epistatic to Ptch, Rab23, and Smo8; however, the exact role of Tctn3 in the SHH signaling pathway remains elusive. To date, increasing evidence shows that Shh determines cell fate by regulating the PI3K/Akt and mTOR signaling pathways19. Nephrocystin-1 (NPHP1) is an adaptor molecule distributed in the cytoplasm and localized to cilia, cell-cell junctions and cell-matrix adhesion sites21. Both NPHP1 and Tctn3 have been demonstrated to interact with Tctn1 and/or Tctn212. Interestingly, NPHP1 regulates resistance to apoptosis22; however, the elaborate mechanism of this regulation remains largely unknown. Given that both Tctn3 and NPHP1 localize at the TZ of the cilia and some of their interactors are overlapping23, we hypothesized that Tctn3 mutant mice would have abnormal development and increased apoptosis by affecting Shh and PI3K/Akt or mTOR signaling pathways. We further hypothesized that increasing levels of apoptosis would probably be associated with expression of NPHP1.Previous studies demonstrated that Tctn1, Tctn2, and Tcnt3 act as regulators of the sonic hedgehog signaling (Shh) pathwayIn this study, to examine the function of the Tctn3 protein in CNS development and its effect on cell fate, we generated a Tctn3 KO mouse model using the piggyBac (PB) transposon system. We found that Tctn3 KO results in increased apoptosis, abnormal brain development, and prenatal death in mice by altering Shh and PI3K/Akt signaling pathways.24. The mice were produced by conventional pronuclei injection of linear plasmids to ensure co-integration of both donor and helper plasmids in the same site. Double positive male offspring were mated with wild-type mice to generate transposase-negative mutants that carry remobilized PB insertions. The Tctn3 mutant line was one of the mutagenesis lines generated in the offspring. The mutation was mapped by inverse PCR as described25. Experimental mice were raised in specific pathogen-free animal housing at the experimental animal center of Soochow University. All mice were placed in cages with a maximum of five mice and 12 h of light (7 am to 7 pm) /dark (7 pm to 7 am) cycle. Behavioral tests were performed at the light cycle with illumination background of 120 lux. All procedures and protocols in this study were approved by the Institutional Animal Care and Use Committee of Soochow University and followed the Guidelines for the Care and Use of the National Institutes of Health (NIH).Tctn3 mutant mice were generated by insertional mutagenesis in the C57 background. Mice carrying single-copy PB transposon PB [Act-RFP] in the genome were mated with mice carrying transposase Act-PBase26. Protein signals were visualized using the ECL chemiluminescence system  and detected on medical X-ray films. Films were scanned, and densitometric analysis of the bands was performed with AlphaEase Image Analysis Software . Primary antibodies were rabbit polyclonal NeuN ; rabbit polyclonal anti-phospho-Akt (Ser473) , anti-mTOR  and anti-phospho-mTOR (Ser2448)  ; rabbit polyclonal anti-Akt ; rabbit polyclonal anti-NPHP1  and rabbit polyclonal anti-(ADP-ribose) polymerase-1 ; rabbit polyclonal anti-Bax ; rabbit monoclonal anti-Bcl-2 ; mouse monoclonal anti-tubulin (T8328 1:20000) ; and mouse monoclonal anti-GAPDH .Mouse brains at embryonic day 12.5 (E12.5) were collected and homogenized utilizing lysis buffer containing protease inhibitor cocktails. After homogenates were centrifuged at 4 °C, supernatants were collected, and protein concentrations were determined. Samples (30–50 μg) in sample buffer were separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Subsequently, proteins were transferred to membranes , and membranes were blocked with 5% milk in phosphate-buffered saline/0.1% Tween20 (PBST) for 1 h to remove non-specific binding. Membrane blots were incubated with primary antibodies overnight with shaking at 4 °C. After washing, membrane blots were incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. The method of detection of immunoreactive proteins was performed as previously described27. Real-time PCR was performed in a 20 μl volume using a 7500 real-time PCR machine . For a 20 μl reaction, 50 ng of single-stranded cDNA was used. The following primers were used: Gli1 forward primer, 5′-CCAAGCCAACTTTATGTCAGGG-3′, Gli1 reverse primer, 5′-AGCCCGCTTCTTTGTTAATTTGA-3′; Ptch1 forward primer, 5′-AAAGAACTGCGGCAAGTTTTTG-3′, Ptch1 reverse primer, 5′-CTTCTCCTATCTTCTGACGGGT-3′; Tctn3 forward primer, 5′-ATCGCAGTCCTTGTCACAGCAG-3′, Tctn3 reverse primer, 5′-CAGCTCCTCCGGATTCGTCTG-3′; GAPDH forward primer, 5′-CATGGCCTTCCGTGTTCCTA-3′, GAPDH reverse primer, 5′- CTTCACCACCTTCTTGATGTCATC-3′. GAPDH was used as an internal control. The relative ratio of mRNA was calculated using the 2−ΔΔCt method. Each sample was run in triplicate, and the average value was calculated.Total RNA was extracted from mouse brains at E12.5 with an RNeasy Plus Mini kit according to the instructions. Reverse transcription was performed using a Transcriptor First Strand cDNA synthesis Kit  as described previouslyFor immunofluorescent staining, E10.5 embryonic brains were fixed in 4% paraformaldehyde. After dehydration in 30% sucrose, the embryonic brains were cut into sections with a frozen cryostat at −20 °C. Frozen brain slices were mounted on glass. After blocking with PBS containing 0.1% Triton X-100 and 1% heat-inactivated goat or sheep serum for 10 min, the slices were incubated with primary antibodies at 4 °C overnight. On the second day, sections were washed with PBS and then incubated with secondary antibodies at room temperature for 1 h. Images were digitized under a fluorescence microscope  with a camera. The primary antibodies used in this study as were as follows: Olig2 ; Shh, Foxa2, Nkx2.2, Hb9, Isl1/2, Pax6, and Pax3 ; and Arl13b .g for 30 min. The protein concentration of supernatants was determined. Samples containing 500 μg protein in 500 μl were pre-absorbed with protein A agarose beads  for 1 h at 4 °C with gentle shaking. After centrifuging, the supernatants were incubated with 5 μl rabbit polyclonal anti-Tctn3  antibody or control rabbit IgG at 4 °C overnight. On the second day, Protein A beads (50 μl) were added and incubated for 2 h at room temperature. Further, samples were centrifuged for 2 min at 1000 × g at 4 °C. Beads were further washed with PBS three times. The bound proteins were eluted with sample buffer containing 100 mM Tis-HCl (pH 6.8), 4% sodium dodecyl sulfate, 0.2% bromochlorophenol blue, 200 mM dithiothreitol, and 20% glycerol and were heated at 96 °C for 10 min. Eluates were collected for Western blot analysis.Fresh brain tissues were homogenized in lysis buffer, and homogenates were centrifuged at 16,000 × 2. When cells reached 50–60% confluence, they were incubated with DMSO/SC79 (4 μg/ml) for 24 h for apoptosis detection29.Tctn3 Het and KO mouse pMEFs were prepared from E12.5 mouse embryos. The pMEFs were cultured in DMEM supplemented with 10% FBS , 1% penicillin-streptomycin at 37 °C and 5% CO30. After cells were stained with Annexin V-FITC/PI, flow cytometric analysis was performed using a Becton Dickinson FACS Calibur flow cytometer . Data were analyzed with Cell Quest Pro software (BD Bioscience).The pMEFs were incubated with DMSO/SC79 (4 μg/ml) for 24 h. Apoptotic cells were identified using the Annexin V-FITC apoptosis kit 31. After removing skin and viscera, newborn mice were fixed in 95% ethanol for 5–7 days. Mice were put into alcian blue solution  and left for 1–2 days. Next, mice were washed with 95% ethanol for another 1 day and washed with a freshly prepared 1% KOH solution until mice appeared quite clear. Embryos were placed in alizarin red S/KOH solution  until bone was stained purple. The bones were rinsed in a series of mixtures of 2% KOH and glycerol solution  and swirled occasionally for several weeks. Specimens were stored in 100% glycerol.Skeletal staining with alizarin red S and alcian blue was performed as described before33.The elevated plus-maze was approximately 70 cm above the ground. The maze consists of two opposite open arms (30 cm × 5 cm) and two opposite closed arms that have 14-cm-high opaque walls. At the beginning of the experiment, animals were placed at the center of the plus-maze facing open arms, and their behaviors were recorded for 10 min. After each test, the maze was cleaned with 50% ethanol solution to eliminate mouse odor. The time spent in closed arms and open arms was recorded as previously described3) consists of white plastic opaque walls. The mice were placed in the center of the field. Their movements in the field were recorded over a 6-min session. Central entries and the total time spent in the central area (20 × 20 cm2) were recorded as previously described33.The open field device  to a 14 cm depth. Immobility time was calculated as the period during which they stopped struggling. Time spent floating on the water and smallest movement to keep floating on water were also counted. The test was recorded for 6 min.The forced swim test was performed as previously described36 with slight modifications. Mouse tails were taped to the edge of a shelf and suspended 35 cm above the desktop. The immobility time, which excludes the time spent struggling, was recorded using a stopwatch for 6 min.The tail suspension test was carried out according to previous reports26.The rotarod apparatus  can reflect coordination performance. The mice were trained for three 5-min trials at 5 rpm on two subsequent days, and the test was carried out on the third day. During the test, the rotating rod was set to speed up from 0 to 40 rpm over 5 min. The latency to fall from the rotarod was averaged over three trialst test was used to determine the differences between two groups (factor: genotype). The differences among groups were determined with two-way ANOVA followed by Bonferroni post hoc tests (factors: mouse genotype and treatment). P values < 0.05 were considered statistically significant.All data are expressed as mean ± SEM. GraphPad Prism 5  was used for statistical analysis. Student’s 12. Furthermore, Tctn3 KO mice exhibited dome-shaped heads with a hole, decreased anteroposterior length  mice were generated as described above. Heterozygous Tctn3 mice were crossed to produce Tctn3 KO mice. In all surviving neonates, only 2 Tctn3 KO mice were found but were dead and subsequently used for bone staining. This suggested that Tctn3 KO causes prenatal death. To further explore this, we collected whole embryos at E10.5 and extracted total RNA. DNA samples were also extracted to verify the genotypes. Our results showed that Tctn3 mRNA was not detected in Tctn3 KO mice; however, relative levels of Tctn1 and Tctn2 mRNA were almost unchanged in Tctn3 KO mice Fig. . These rgth Fig.  and abnogth Fig. . Since h8. Tctn1 and Tctn2 were found to play an important role in neural tube patterning by mediating the hedgehog pathway12. To determine if Tctn3 regulates neural tube patterning as well, we examined neural tube patterning-related proteins in Tctn3 KO embryos. The expression of Shh in the ventral neural tube was significantly decreased in Tctn3 KO embryos. Consistent with Shh expression in Tctn3 mutant mice, Foxa2, which is normally induced by a high level of Shh was also lost in the Tctn3 KO embryos. The expression domain of Nkx2.2, which was positive V3 progenitors specified, shifted ventrally. Olig2, known for determining motor neuron and oligodendrocyte differentiation, shifted ventrally. The patterning and expression of Hb9 or Isl1/2, the motor neuron marker, was affected in Tctn3 KO mice as well. Pax6 expression was also altered, slightly shifting to the ventral side. However, the expression of Pax3 in the dorsal neural tube, which is ventrally limited by low Shh signaling, was unchanged. In addition, Arl13b, a cilia-specific marker, was significantly reduced , total Akt (t-Akt), phosphorylated-mTOR (p-mTOR), and total mTOR (t-mTOR) in the brains of Tctn3 KO mice. Compared with Tctn3 Het KO mice, p-Akt was significantly decreased in the brains of Tctn3 KO mice (P < 0.05, Fig. P > 0.05, Fig. P > 0.05, Fig. Shh signals are considered to regulate cell fate through PI3K/Akt and/or mTOR signaling pathways10, we further examined whether knockout of Tctn3 also affects apoptosis of pMEFs. We cultured pMEFs of Tctn3 het and KO mice at E12.5 to explore whether presence of apoptosis in the MEFS of Tctn3 KO mice. Consistent with the presence of elevated apoptosis in the brains of Tctn3 KO mice, Tctn3 KO led to increased levels of apoptotic pMEFs from Tctn3 KO mice as detected by flow cytometry (Fig. 28, reduced levels of apoptotic pMEFs (P < 0.05, Fig. P < 0.05, Fig. P > 0.05, Fig. P < 0.05, Fig. P < 0.05, Fig. P < 0.05, Fig. As Tctn proteins are crucial for ciliogenesis in pMEFstry Fig. . More in42. NPHP1 deficiency or mutation also causes ciliopathies such as JBTS, NPHP or BBS48. Previous studies have demonstrated that both Tctn3 and NPHP1 interact with Tctn1 and/or Tctn212. Intriguingly, NPHP1 also regulates resistance to apoptosis but not the cell cycle22. To ascertain whether NPHP1 expression is altered in the brains of Tctn3 KO mice, we collected brain tissues from E12.5 mice to examine the protein expression of NPHP1 in Tctn3 KO mice. Our results showed that NPHP1 expression in the brains of Tctn3 KO mice was significantly decreased compared with that in Tctn3 Het KO mice (P < 0.05, Fig. NPHP1 is a cytoplasmic adaptor molecule that localizes to cilia, cell-cell junctions, and cell-matrix adhesion sites9. The Shh signaling pathway is one of the most important developmental mediators1438. Although tectonic proteins regulate the Hedgehog signaling pathway12 and its downstream signals such as Gli1 and Ptch1, the three tectonic proteins still display different roles in mediating the Shh pathway and development in mice. Tctn1 null mice present with heterotaxia, holoprosencephaly, microphthalmia, and hind limb polydactyly, similar to the phenotypes of mice with Shh signaling defects8. Apart from these characteristics of Tctn1 mice, Tctn2−/− mice present additional phenotypes, including cleft palate, right-sided stomach, and ventricular septal defect12. In our study, Tctn3 mutant embryos display edema on the back of the upper body and randomized heart looping9, as well as polydactyly (Fig. 49. Based on the phenotype of mutant mice, we speculate that all three members of the Tctn family have various functions in development and regulation of the Shh signaling pathway.The tectonic protein family is a group of TZ proteins, including Tctn1, Tctn2, and Tctn3, which comprise one membrane binding protein and two single-pass membrane proteinsyly Fig. , abnormayly Fig.  and abnoyly Fig. . The pol12. Tctn1−/− and Tctn2−/− mice present with significantly decreased Islet1/2-positive motor neurons, while Pax6, a molecule suppressed by Shh, is widely distributed12. In this study, although Tctn3 KO mice exhibit normal Pax3 patterning in the dorsal neural tube, Shh, Foxa2, Nkx2.2, Olig2, Hb9, Islet1/2, and Pax6 patterning are significantly ventrally shifted. In addition, the Arl13b protein, a marker of cellular cilia, was significantly decreased in Tctn3 KO mice (Fig. 9 found that the Shh signal pathway was disrupted in Tctn3 null mice, as shown by undetectable Foxa2 and ventrally shifted Nkx2.2; however, Pax6, Pax7, and Hb9 distributions were normal, which was different from our results. Therefore, our Tctn3 KO mice seemed to have more severe symptoms. For example, these mice had prenatal lethality, microphthalmia, and bone changes. This inconsistency may be explained by different approaches to create the Tctn3 KO. In the previous report, mutant Tctn3 mice were produced by deleting the exon 3 of Tctn3 with a Cre-Loxp system. In the present study, Tctn3 mutant mice were generated by insertional mutagenesis. In summary, we conclude that Tectonic proteins play vital roles in neural tube development and the Shh signaling pathway.In addition, several molecular markers regulated by the Shh signal were changed in Tectonic mutant mouse embryos9, the role of Tctn3 in the Shh signaling pathway and development is distinct from that of Tctn1 and Tctn2. This raises an interesting question as to whether Tctn3 KO mice have other phenotypes. Increasing evidence indicates that the Shh signaling pathway plays a significant role in inhibiting apoptosis18. The decline in the Shh signaling pathway (Fig. Although the three Tctn proteins have high levels of homologyway Fig.  in the bway Fig.  and the way Fig. . These r41. In this study, p-Akt was significantly decreased in the brains of Tctn3 KO mice, but p-mTOR was not changed (Fig. 12. Interestingly, NPHP1 can also regulate resistance to apoptosis but not the cell cycle in mammals22. Protein expression of NPHP1 was also significantly decreased in the brains of Tctn3 KO mice (Fig. Many studies have shown that the Shh signaling pathway is involved in the regulation of cell fate through PI3K/Akt and/or mTOR signaling pathwaysged Fig. , b. In aice Fig. , and NPHice Fig. . These r9. The pMEFs from Tctn3 KO mice exhibited an increase in apoptosis, as detected by flow cytometry analysis (Fig. 28, elevated levels of p-Akt in pMEFs from Tctn3 KO mice (Fig. To explore whether increased apoptosis in Tctn3 KO mice could be prevented by Akt agonists, we cultured pMEFs, in which ciliogenesis is regulated by Tctn3 at E12.5sis Fig. . Use of ice Fig.  and reduice Fig. . These r3. As one of the important cilial network proteins, Tectonic mutations are also known to cause different ciliopathies, including MKS, OFDS, and JBTS52. These ciliopathies are developmental diseases that follow a recessive pattern, with no symptoms found in carriers of the mutation, which is consistent our Tctn3 het KO mouse data (Figs. 10. At the same time, Tectonic proteins are connected with components of some important signal pathways, such as the Shh signaling pathway and apoptosis-related signaling pathways, as evidenced in this study. Different mutations in Tectonic genes may cause loss or gain of functions resulting in different phenotypes and heterogeneity in ciliopathies53. However, the underlying mechanisms of connections between Tectonic proteins and different important signaling pathways remain unclear. Understanding the molecular pathogenesis of Tectonic-induced ciliopathies may help treat patients with ciliopathies and improve their quality of life.Destruction of the integrity of ciliated protein complexes leads to defects in the cilium membrane protein components, leading to many human ciliopathiesta Figs. –3. As stIn summary, Tctn3 deficiency disrupts the Shh signaling pathway and the neural tube patterning, resulting in abnormal embryonic development and prenatal death in mice. Therefore, Tctn3 has distinct roles in the normal development of mouse embryos including brains, eyes, and bones that could not be compensated by Tctn1 and Tctn2 proteins.Table S1Figure S1Figure S2Figure S3Figure S4Supplementary figure legends


Drosophila, the activity of the TGFβ/Activin signalling branch has been linked to the regulation of cell growth and proliferation, but the cellular and molecular basis for these functions are not fully understood. In this study, we show that both the RII receptor Punt (Put) and the R-Smad Smad2 are strongly required for cell and tissue growth. Knocking down the expression of Put or Smad2 in salivary glands causes alterations in nucleolar structure and functions. Cells with decreased TGFβ/Activin signalling accumulate intermediate pre-rRNA transcripts containing internal transcribed spacer 1 regions accompanied by the nucleolar retention of ribosomal proteins. Thus, our results show that TGFβ/Activin signalling is required for ribosomal biogenesis, a key aspect of cellular growth control. Importantly, overexpression of Put enhanced cell growth induced by Drosophila Myc, a well-characterized inducer of nucleolar hypertrophy and ribosome biogenesis.Signalling by TGFβ superfamily factors plays an important role in tissue growth and cell proliferation. In  Transforming growth factor beta (TGFβ) and bone morphogenetic protein (BMP) signalling pathways play multiple roles during animal development through the regulation of cellular growth, proliferation, differentiation and survival . At the Drosophila, where both the BMP and TGFβ/Activin branches are crucial regulators of developmental processes [The TGFβ pathway is evolutionarily conserved in rocesses . Put is rocesses –11. The rocesses –14, as wrocesses ,9,15.Drosophila TGFβ signalling members  and the nucleolar regulator Viriato (Vito)/Nol12 [In a recent eye-targeted double-RNAi screen, we identified a genetic interaction between several o)/Nol12 . Previouo)/Nol12 . Thus, Vo)/Nol12 . Because2.2.1.putRNAi lines we used (VDRC37279 and NIG-FLY7904R-3) target non-overlapping regions of put and inhibited the progression of photoreceptor differentiation in the eye imaginal disc . This phenotype mimics mutant put phenotypes [put expression in the salivary glands caused a strong reduction in cellular area  with a strong effect on tissue growth (g). In particular, expression of the stronger putRNAi37279 caused a significant decrease in salivary gland area , and completely inhibited the onset of the photoreceptor differentiation . Our recent work established a genetic interaction between put and vito, which encodes for a regulator of nucleolar organization and tissue growth [figure 1d–f, and not shown). Knocking down put caused an expansion of the nucleolar area, reflected in an increased ratio between nucleolar and nuclear areas. Expression of putRNAi7904R-3 resulted in a 62% increase in the nucleolar/nuclear area ratio (p < 1 × 10−4), whereas expression of putRNAi37279 caused an even stronger increase . Thus, for put depletion in salivary glands, we observe a direct correlation between increased nucleolar/nuclear ratio and diminished tissue growth (R2 = 0.99468). This suggests that put controls nucleolar dynamics during salivary gland cell growth. Importantly, the weaker putRNAi7904R−3 did not significantly affect nuclear size (figure 1j), suggesting that the reduced nuclear size caused by the stronger putRNAi37279 (figure 1j) is secondary or a consequence of the observed nucleolar alterations.To study possible cell-autonomous functions of TGFβ signalling in salivary gland cell growth, we downregulated the expression of the RII receptor Put . RNA intenotypes , and conlar area a–c,h wite growth g. In pare growth . Becausee growth ,20, we esmad2RNAi affected growth of the eye disc  [madRNAi strongly interfered with both tissue growth and patterning  [smad2RNAi caused a strong increase in the nucleolar/nuclear area ratio in salivary gland cells, which is not induced by madRNAi expression (f). Furthermore, co-expression of madRNAi failed to enhance the increase in nucleolar size, or the decrease in cellular and tissue size, induced by smad2RNAi (g–j). In addition, salivary glands of larvae mutant for put or for babo, the Activin branch-specific RI receptor, also displayed a nucleolar phenotype, not observed in mutants for tkv, the BMP branch-specific RI receptor (a–f). Taken together, these results show that the TGFβ/Activin pathway is required for growth and normal nucleolar dynamics.The RII receptor Put heterodimerizes with RI receptors Tkv or Babo to mediate BMP and TGFβ/Activin signalling, respectively. To study the contribution of each signalling branch to nucleolar regulation, we knocked down the expression of the TGFβ/Activin-branch-specific R-SMAD, Smad2 ,21, and ure S1e) , whereasure S1f) . Importapression f. Furthereceptor a–f. Take2.2.a), as previously observed [putRNAi caused a strong nucleolar accumulation of RpL41 . Similarly, we also observed an increase in the nucleolar localization of Vito (figure 4d–f). Nucleolar enrichment of Vito was not homogeneous, as we detected intranucleolar regions with higher Vito levels (figure 4d–f). These results prompted us to further evaluate the role of TGFβ/Activin signalling in nucleolar structure and ribosome biogenesis. In control cells, immunostaining with αRpL22, αRpL10A and αRpS6 antibodies showed that these ribosomal proteins are mainly cytoplasmic, possibly reflecting a transient association with pre-ribosome subunits at the nucleolus . Interestingly, in putRNAi both RpL22 and Rpl10A are concentrated in the peripheral nucleoplasm and in the nucleolus (b–b″), whereas RpS6 accumulates in granular intranucleolar spots (d-d′). In comparison with control cells, RpL11 is also found at higher levels in these nucleolar granular spots, where it co-localizes with RpS6 (d″). This pattern of nucleolar accumulation was not a general attribute of all ribosomal proteins. RpS9 is mainly cytoplasmic in control cells and its levels decrease in putRNAi without any evidence of nucleolar re-localization .The increased size ratio of the nucleolus apparently contradicts the reduction in tissue growth observed when the TGFβ/Activin pathway is attenuated. One possibility is that the increase in size is reflecting a defective, rather than a gain of nucleolar function (e.g. a defective production of ribosomes). The nucleolus plays a major role in cell growth through the coordination of three steps in ribosome biogenesis: transcription of pre-rRNA by polymerase I, processing of pre-rRNA, and assembly of the large (60S) and small (40S) ribosome subunits . Defectsfigure 4observed . Inhibitucleolus a–a″,c–c″ucleolus b–b″, whear spots d-d′. In ith RpS6 d″. This putRNAi or smad2RNAi expression confirmed the presence of nucleolar hypertrophy in these genotypes (e–g). Further, low-contrast intranucleolar regions could be observed, although it is unclear if these regions correspond to the accumulation spots for Vito and RpS6 observed using confocal microscopy (figures f and 5d). Moreover, we detected an accumulation of densely packed particles in the nucleoplasm when compared with control cells . Importantly, only individual particles were found occasionally in controls (figure 5e″) and are never found as clusters in our control samples . The size of these particles was on the scale expected for pre-ribosomal intermediates undergoing maturation in the path from the nucleolus to the cytoplasm [Drosophila midgut cells by knockdown of Nopp140 [a) [a) [putRNAi and smad2RNAi cells, we detected a strong accumulation of uncleaved pre-rRNA intermediates containing the ITS1 region (b). To further distinguish whether this accumulation is derived from an increased transcription or an accumulation of the uncleaved pre-RNA intermediates, we quantified the relative abundance of pre-rRNA transcripts containing the external transcribed spacer (ETS) region. ETS-containing transcripts are short-lived, as the ETS is the first region to be processed with fast kinetics, and can be used as a proxy for the pre-rRNA transcription rate by the RNA polymerase I. Thus, when the levels of ITS1 were normalized to ETS levels, both TGFβ RNAis present about a threefold increase of ITS1-containing intermediate precursors. Furthermore, the levels of the small ribosome subunit 18S rRNA were also significantly reduced in these cells, whereas no significant differences were detected for the 28S rRNA (b). Together, these results point towards stalled ribosome biogenesis.Ultrastructural TEM analysis of salivary gland cells where TGFβ/Activin signalling was inhibited by either enotypes e–g. Furt figures f and 5d.ytoplasm . These p Nopp140 . Thus, op140 [a) . After b [a) [a) ,31. Inte1 region b. To fur28S rRNA b. Togeth2.3.Drosophila cells, this mechanism requires coordination between nucleolar hypertrophy and the stimulation of pre-rRNA transcription and processing [figure 7a,b,d,e,g,h) [figure 7c,f,g,h). The overexpression of Put, on its own, was not sufficient to induce growth in the salivary glands (not shown). In support of these observations, in the eye imaginal disc and resulting adult retinas, the overexpression of Put was also able to synergize with Myc, increasing overall tissue size . These results suggest that TGFβ signalling cooperates with dMyc to control nucleolar function and mass accumulation.The ability of Myc to increase ribosome synthesis is an essential mechanism by which Myc promotes both cell growth and proliferation, as well as tumorigenesis . In bothocessing ,18,33–36d,e,g,h) ,37. dMycd,e,g,h) . It has d,e,g,h) . Remarka3.Drosophila larval salivary gland. In this simple tissue model, cell growth control can be untangled from cell proliferation and ligand gradient control. Previously, the Activin RI receptor Babo and Smad2 were shown to be specifically required for cellular proliferation and overall growth of the wing imaginal disc [Drosophila [vito, a nucleolar regulator of growth [Taken together, our results show that members of the Activin branch of the TGFβ signalling pathway (the RII receptor Put and the R-Smad Smad2) are autonomously required for cell and tissue growth in the nal disc . Interesnal disc . Recentlosophila , and to osophila . These rf growth , lead usf growth . When wef growth ,43, thusCould TGFβ regulate ribosome biogenesis through other novel mechanisms? In fact, in breast cancer cells, a cancer-specific nuclear translocation of TβRI was shown to regulate nuclear mRNA processing . Further4.4.1.lacZ and the wild-type strain w1118. UAS-CD4tdTomato was used to report salivary glands expression of ey-Gal4 . AB1-Gal4 and ptc-Gal4 were used as salivary gland alternative drivers with similar results to ey-Gal4 . The TGFβ RNAis were obtained from different collections: putRNAi#37279 (VDRC), putRNAi 7904R-3 (Nigfly), smad2RNAi  and madRNAi . The following TGFβ pathway mutants were obtained from the Bloomington Stock Center: put135, put10460, babo32, baboK16912, tkv1 and tkv8. Overexpression studies were done using UAS-Put [All crosses were raised at 25°C under standard conditions and for synchronization; all the conditions were analysed after a single day of egg collection. The following stocks  were used: ey-Gal4, UAS- UAS-Put  and UAS- UAS-Put . The pro4.2.g–j′).Eye-antennal imaginal discs and salivary glands were prepared for immunohistochemistry using standard protocols. As the growth conditions strongly affect salivary gland size, all the experiments were controlled by synchronization of L3 wandering larvae after a single-day egg collection. To further control this issue, a controlled the egg laying for 5 h was set up, and the salivary glands were analysed 96 h after egg laying , mouse anti-Dlg at 1 : 1000 , rabbit anti-Viriato (Vito) at 1 : 250 (ABGent), rat anti-DCad at 1 : 100, mouse anti-AH6 at 1 : 10 (DSHB), rabbit anti-Fibrillarin at 1 : 250 , mouse anti-Fibrillarin at 1 : 500 , mouse anti-RpS6 at 1 : 100 , mouse anti-RpL11 at 1 : 100 , mouse anti-RpL10A at 1 : 400 , rabbit anti-RpL22 at 1 : 100 (kind gift from Dr Vassie Ware). To stain for cellular limits phalloidin conjugated with rhodamine was used at a dilution of 1 : 1000. Appropriate Alexa-Fluor conjugated secondary antibodies were from Molecular Probes. Images were obtained with the Leica SP2 confocal system or Leica SP5 confocal system and processed with Adobe P4.3.mageJ 1.48r software , considering the limits stained by Arm, Dcad or RhPh and represented as arbitrary units. The cellular parameters shown in this study were measured using the Polygon selection tool of ImageJ 1.48r. The nucleolar area was determined using the nucleolar markers RpL41YFP, anti-AH6 or anti-Fibrillarin and DAPI staining was used to stain for the nuclear area. The results are presented as the ratio of the nucleolar area to the nucleus that it corresponds to. The intensity of the nucleolar components was determined using a fixed ROI circle, and the mean intensity of each nucleolus was measured using ImageJ. To each measurement, another nucleolar component was used as reference . For each genotype, five to six nuclei from the proximal region of at least five to six independent salivary glands were used. Statistical analysis and generation of the graphical output was done using GraphPad Prism v. 5.0. Statistical significance was determined using an unpaired, two-tailed Student's t-test, with a 95% confidence interval, after assessing the normality distribution of the data with D'Agostino–Pearson normality test.Salivary gland areas were measured using the Polygon selection tool of I4.4.Dissected third-instar salivary glands were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for 30 min and post-fixed with 4% osmium tetroxide. After washing, salivary glands were incubated with 0.5% uranyl acetate (30 min) and further dehydrated through a graded ethanol series . Salivary glands were then soaked in propylene oxide for 10 min and then in a mixture (1 : 1) of propylene oxide and Epon resin (TAAB Laboratories) for 30 min. This mixture was then replaced by 100% Epon resin for 24 h. Finally, fresh Epon replaced the Epon and polymerization took place at 60°C for 48 h. Ultrathin sections were obtained using an ultramicrotome, collected in copper grids and then double contrasted with uranyl acetate and lead citrate. In total, at least 16 independent cells of five independent salivary glands were analysed for each genotype. Micrographs were taken using a TEM Jeol JEM-1400, with Orius SC 1000 digital camera (80 kV).4.5.putRNAi37279 50–60 salivary glands were dissected and 40–50 salivary glands were dissected for ey-Gal4; smad2RNAi2262R−2).For qPCR experiments, all the RNAis were induced with the ey-Gal4 driver, and the salivary glands of wandering L3 instar larvae were dissected. The number of salivary glands was determined according to its size to yield similar RNA concentrations  according to the manufacturer's instructions and treated with Turbo DNase I (Ambion). cDNA was generated by reverse transcription with the SuperScript III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen). Quantitative real-time PCR analysis was performed in triplicate in 20 µl reactions containing iQ SYBR Green Supermix (BioRad), each gene-specific primer at 250 nM and 1 µl of cDNA template. Cycling conditions in a BioRad iQ5 instrument were 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 10 s and annealing for 30 s at 53°C, 60°C or 64°C depending on the primer set. Fold change relative to the expression of pression , was calCaMKII (control): Fw—TTACACCATCCCAACATAGTGCRev—CAAGGTCAAAAACAAGGTAGTGATAG;28S: Fw—GGAGGATCTTCGATCACCTGATGRev—GCTGCTCAACCACTTACAACAC;18S: Fw— TGGTCTTGTACCGACGACAGRev—GCTGCCTTCCTTAGATGTGG;ITS1: Fw—TTATTGAAGGAATTGATATATGCCRev—ATGAGCCGAGTGATCCAC;ETS: Fw—GCTCCGCGGATAATAGGAATRev—ATATTTGCCTGCCACCAAAA.


Pre-treatment with TPEN increased simvastatin-induced cytotoxicity and eliminated the distinction between both cells of simvastatin-induced cytotoxicity. These results suggest that Zn deficiencies may be involved in the increased risk for muscle complaints in ESKD patients. In conclusion, the increased severity of statin-induced rhabdomyolysis in ESKD patients may be partly due to trace elements deficiencies.Patients with end-stage kidney disease (ESKD) are at higher risk for rhabdomyolysis induced by statin than patients with normal kidney function. Previously, we showed that this increase in the severity of statin-induced rhabdomyolysis was partly due to uremic toxins. However, changes in the quantity of various trace elements in ESKD patients likely contribute as well. The purpose of this study is to determine the effect of trace elements on statin-induced toxicity in rhabdomyosarcoma cells exposed to uremic serum (US cells) for a long time. Cell viability, apoptosis, mRNA expression, and intracellular trace elements were assessed by viability assays, flow cytometry, real-time RT-PCR, and ICP-MS, respectively. US cells exhibited greater simvastatin-induced cytotoxicity than cells long-time exposed with normal serum (NS cells) . Intracellular levels of Mg, Mn, Cu, and Zn were significantly less in US cells compared to that in NS cells ( HowThere is an imbalance of various trace elements, such as low plasma concentrations of Zn, in ESKD patients. In addition, patients undergoing hemodialysis (HD patients) have abnormally high plasma Cu/Zn ratios, low activity of superoxide dismutase (SOD), and high levels of oxidative stress; Zn supplementation in HD patients ameliorates Cu/Zn ratios and may reduce oxidative stress . AtorvasThe aim of this study is to determine whether variation in the concentration of trace elements affects statin-induced cytotoxicity. Therefore, we investigated the effects of Mg, Ca, Mn, Cu, and Zn on statin-induced cytotoxicity in long-time uremic serum (US)-exposed human rhabdomyosarcoma (RD) cells.50 value of simvastatin treated US cells was significantly decreased compared to that of normal serum (NS) cells  cells . In contNS cells .The number of apoptotic cells in US and NS cells was not different in the non-treatment condition, but apoptosis in US cells treated with simvastatin was greater than that in NS cells .The intracellular concentrations of Ca in NS and US cells were not different, whereas the intracellular concentrations of Mg, Mn, Cu, and Zn were significantly lower in US cells than in NS cells .N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine) compared to untreated controls  1A was significantly lower in US cells than in NS cells. In contrast, the mRNA level of SOD1, MT2A, and glutathione peroxidase 1 (GPx1) were not significantly different in US and NS cells .We had previously shown that statin-induced cytotoxicity in differentiated RD cells was augmented by pre-treatment with uremic toxins—hippuric acid, 3-carboxy-4-methyl-5-propyl-2-furanpropionate, indole-3-acetic acid, and 3-indoxyl sulfate, whereas cisplatin-induced cytotoxicity was not affected by pre-treatment with uremic toxins . In thisOne factor that may influence sensitivity to specific drugs in ESKD patients is an imbalance of various trace elements. Intracellular concentrations of Mg, Mn, Cu, and Zn were significantly lower in US cells than in NS cells . Ari et Pre-treatment with the membrane-permeable heavy metal chelator TPEN significantly decreased intracellular concentrations of Mg, Cu, and Zn in both US and NS cells  and incrmRNA levels of SOD1, MT2A, and GPx1 in US cells were comparable to that in NS cells, but the level of SOD2 mRNA in US cells was significantly higher than that in NS cells . In addimRNA expression of MT1A in US cells was significantly lower than in NS cells . MetalloThe limitations of this study include lack of changes in oxidative stress, and the relationship between protein and mRNA expression levels. Accordingly, typical markers of oxidative stress or ROS production and the protein expression of antioxidant enzymes or a metal transporter need to be measured directly to confirm our findings.In this study, we showed that the augmentation of simvastatin-induced cytotoxicity in US cells was caused, in part, by promotion of apoptosis, which resulted from trace element deficiencies such as Zn. The simvastatin-induced cytotoxicity may be alleviated partially by supplementation of trace elements such as Zn. However, further research is needed to confirm this mechanism and effect of variation in other trace elements. Our findings contribute to a growing body of evidence explaining why statin-induced rhabdomyolysis is enhanced in ESKD patients and may lead to more appropriate use of statins in these patients in the future.Simvastatin was purchased from Toronto Research Chemicals, Inc. . Losartan was purchased from LKT Laboratories, Inc. . Cisplatin was purchased from Wako Pure Chemical Industries . TPEN was purchased from Santa Cruz Biotechnology, Inc. . Annexin V-fluorescein isothiocyante (FITC) was purchased from BioLegend, Inc. . The Cell Quanti-Blue™ Cell Viability Assay Kit (CellQuanti-Blue™) was purchased from BioAssay Systems . Pooled human serum as NS was purchased from Merck Millipore Co. . Serum that was pooled from more than 400 dialysis patients as US was obtained from Shirasagi Hospital . Because the patients were administered various drugs for medication, those drugs might affect our research. All uremic sera, however, were collected from patients just before hemodialysis. Because HD patients do not administer drugs before hemodialysis in general except for prophylactic drug for adverse events such as dialysis hypotension, it was thought that there were negligible effects of prescription drugs in US. This study  was approved in advance by the Shirasagi Hospital and Kyoto Pharmaceutical University ethical review board on 11 December 2008 and 1 April 2008, respectively.2. During this period, RD cells were re-plated at a density of 1 × 106 cells per 10 mL in media every third or fourth day. The resulting cells were named NS and US cells, respectively.The RD cell line was purchased from American Type Culture Collection . Dulbecco’s modified Eagle’s medium  without fetal bovine serum  contained sodium hydrogen carbonate (3.7 g), 100 U/mL penicillin, and 100 µg/mL streptomycin and 0.1 mM non-essential amino acids solution . NS and US were deproteinized using three volumes methanol and the supernatants were dried under a stream of nitrogen at 50 °C. The residue was dissolved in 10 volumes DMEM without FBS and ultrafiltered using a 0.22 µm membrane filter. The final concentration of 10% FBS was ultrafiltered in DMEM with serum using a 0.45 µm membrane filter, and these media were named 10% NS medium or 10% US medium, respectively. RD cells were exposed for at least one month to 10% NS medium or 10% US medium at 37 °C with 5% CO3 cells/well/100 µL into 96-well plates in DMEM with 10% FBS for three days at 37 °C with 5% CO2. These cells were then cultured in DMEM with 1% FBS (differentiation medium) for seven days to induce differentiation. In the pre-treatment experiment with TPEN, 1 µM TPEN was added to the differentiation medium. After removal of the medium from differentiated RD cells, differentiated RD cells were exposed to medium containing the following test compounds for three days at 37 °C with 5% CO2. The concentrations of simvastatin, losartan, and cisplatin (test compounds) used were 0.03125–32 or 0.25–256 µM, 16–4096 µM, and 0.5–512 µM, respectively.RD cells that grown as the following are a model cells of human skeletal muscle and used for myotoxicity evaluation in this study . NS and After treatment with the test compounds for three days, cell viability was measured using CellQuanti-Blue™ and a microplate reader .50) value was calculated using the non-linear least squares program (MULTI)L, Lmax, C, and γ are cell viability (% of control), maximum cell lethality, drug concentration in the medium, and sigmoid function, respectively.The lethal concentration 50 -1-piperazinyl] ethanesulfonic acid (HEPES)/NaOH, pH 7.4). Annexin V-FITC (5 μL) and 50 µg/mL propidium iodide (PI) solution (1 μL) were added to the 100 μL cell suspension and incubated for 15 min in the dark. The reaction solution was diluted five-fold in the same buffer and then analyzed using fluorescence activated cell sorting (FACS) Calibur™ . Apoptotic cells were defined as those that were annexin V-FITC-positive and PI-negative.NS and US cells were incubated in medium containing 8 µM simvastatin for 24 h (shorter than cytotoxicity experiment) at 37 °C with 5% CONS and US cells were lysed by addition of 1% sodium dodecyl sulfate (5 mL). After pouring the cell lysate (4.7 mL) into a nitric acid-treated tall beaker, the lysate was evaporated completely on a hot plate at 170 °C. Upon addition, in turn, of nitric acid, perchloric acid, and hydrogen peroxide to the residue, the residue was dried under the same conditions as described above. Three cycles of the same operation were performed for complete incineration of the sample.r2 > 0.999).The incinerated sample was lysed by addition of nitric acid (9 mL), and the concentrations of intracellular Mg, Ca, Mn, and Zn were determined by inductively coupled plasma-mass spectrometry . Prepared calibration standard samples were 0, 5, 10, 50, 100, and 500 ppb. The overall relationship among the standards was best described by a linear relationship . After adding diethyl pyrocarbonate-treated water (400 µL) to the cell lysates, they were vortex mixed for 15 s, left at room temperature for 15 min, and centrifuged at 12,000× g at 25 °C for 15 min. After adding isopropanol (1 mL) to the supernatant (1 mL), the supernatant was left at room temperature for 10 min. RNA precipitate formed a pellet upon centrifugation at 12,000× g at 25 °C for 15 min. The residue was removed from the supernatant and was washed twice using 75% ethanol (500 µL). After washing, RNase-free water (50 µL) was added to the residue, and the RNA in the residue was quantified spectrophotometrically at 260 nm using a DU®730 (Becton Dickinson). RNA quantity was calculated using the formulaNS and US cells were lysed by addition of RNAzolThe RNA solutions were diluted to 100 µg/mL with RNase-free water and stored at −80 °C.® qPCR . For preparation of cDNA, the total reaction volume per sample was 20 µL . RT-PCR was performed using an i-Cycler iQ  with 37 °C for 15 min (reverse transcription reaction), 98 °C for 5 min (inactivation of reverse transcriptase), and then 4 °C (cooling). DNA was amplified in a total reaction solution volume of 20 µL , 0.4 µL of 10 µM sense primer, 0.4 µL of 10 µM antisense primer, 0.04 µL of 50× ROX reference dye (Toyobo), 2 µL of RT sample). Real-time PCR was performed using a LightCycler® Nano System . The initial denaturation was 95 °C for 1 min and was followed by 45 cycles of amplification, with a thermal cycling profile of 95 °C for 10 s and 60 °C for 30 s. Beta-2-microglobulin (β2M) was used as a housekeeping gene. Primer sequences are specified in Reverse transcriptase polymerase chain reaction (RT-PCR) was performed using ReverTra AceT was calculated using the fluorescence intensity of SYBR® Green I, and the amount of target mRNA relative to β2M mRNA was expressed as 2−⊿CT, where ⊿CT is the value obtained by subtracting the CT value of β2M mRNA from the CT value of the target mRNA.C50 values were expressed as mean ± standard deviation (S.D.) and median , respectively. Non-overlapping confidence intervals of LC50 were considered statistically significant. The significant differences between groups were determined using unpaired Student’s t-test or non-repeated measures analysis of variance (ANOVA) followed by Tukey–Kramer multiple comparison test. A p value less than 0.05 was considered statistically significant.Measured values and LC