Six hPRL variants were identified with different isoelectric point (pI)-relative molecular mass (Mr) distribution on a 2DE pattern, including variants V1 , V2 , V3 , V4 , V5 , and V6 . Compared to controls, except for variants V2-V6 in PRL-adenomas, V2 in FSH+-adenomas, and V3 in NF−-adenomas, the other PRL variants were significantly downregulated in each subtype of pituitary adenomas. Moreover, the pattern of those six PRL variants was significantly different among five subtypes of pituitary adenomas relative to control pituitaries. Different hPRL variants might be involved in different types of PRL receptor-signaling pathways in a given condition. Those findings clearly revealed the existence of six hPRL variants in human pituitaries, and the pattern changes of six hPRL variants among different subtypes of pituitary adenomas, which provide novel clues to further study the functions, and mechanisms of action, of hPRL in human pituitary and in PRL-related diseases, and the potential clinical value in pituitary adenomas.Human prolactin (hPRL) plays multiple roles in growth, metabolism, development, reproduction, and immunoregulation, which is an important protein synthesized in a pituitary. Two-dimensional gel electrophoresis (2DE) is an effective method in identity of protein variants for in-depth insight into functions of that protein. 2DE, 2DE-based PRL-immunoblot, mass spectrometry, and bioinformatics were used to analyze hPRL variants in human normal (control;  Prolactin (PRL) is a four long α-helix protein hormone, which was discovered in mammals in the 1930s by Oscar Riddle, and in humans in the 1970s by Friesen et al. . The hPRpI and Mr. Further studies found that the main source of PRL variants in mammals is not alternative splicing, but cleavage of PRL, and those variants were 14-, 16-, and 22-kDa (n = 3–4) , translocation, re-distribution, and spatial conformation alteration . Normal d 22-kDa . Sohm etd 22-kDa . Althougd 22-kDa . Most ofd 22-kDa –12. Somen = 3–4) . HoweverpI values in the isoelectric focusing (IEF) direction, and different Mr values in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) direction  is an effect method to characterize the isolated PRL variants and identify PTMs with an analysis of amino acid sequence and determination of PTM sites. These types of MS have been used to study the 2DGE-separated pituitary proteins, including matrix-assisted laser desorption/ionization time-of-flight MS (MALDI-TOF MS) peptide fingerprint (PMF), MALDI-TOF-TOF MS and tandem mass spectrometry (MS/MS), and liquid chromatography-electrospray ionization-quadruple-ion trap MS (LC-ESI-Q-IT MS) MS/MS analysis  and the Memphis Regional Medical Center (n = 7), which were approved by University of Tennessee Health Science Center (UTHSC) Internal Review Board (IRB). Human pituitary adenoma tissues were obtained from the Emory University Hospital, which were approved by Emory University Hospital IRB, and Department of Neurosurgery of Xiangya Hospital, which were approved by the Xiangya Hospital Medical Ethics Committee of Central South University, China. Consent was obtained from each patient or the family of control pituitary subject after full explanation of the purpose and nature of all procedures used. All tissues were removed, frozen immediately in liquid nitrogen, and stored at −80°C until processing. The clinical information of pituitary adenoma and control samples is summarized in Table Human control pituitary glands were post-mortem tissues obtained from the National Disease Research Interchange  and 18 cm IPG strip holder on an IPGphor instrument . Before IEF, an aliquot (350 μl) of protein sample solution was loaded onto an IPG strips. The IPG strip was rehydrated about 18 h. IEF was performed at 25°C with the following parameters: a gradient 250 V and 1 h for 125 Vh, a gradient at 1000 V and 1 h for 500 Vh, a gradient at 8000 V and 1 h for 4000 Vh, a step-and-hold at 8000 h and 4 h for 32000 Vh, a step-and-hold at 500 V and 0.5 h for 250 Vh to achieve a total of 36,875 Vh and ~7.5 h analysis. The IPG strip was removed and laid on its plastic back to blot off mineral oil. After IEF, IPG strips were processed with SDS-PAGE.2O), and 50 μl of tetramethylethylenediamine. The IPG strip was equilibrated in 15 ml of reducing equilibration buffer (15 min) that consisted of a trace of bromphenol blue, 375 mmol/L Tris-HCl pH 8.8, 2% w/v SDS, and 20% v/v glycerol. The IPG strip was equilibrated in 15 ml of alkylation equilibrium solution (15 min) that consisted of 2.5% w/v iodoacetamide instead of 2% w/v DTT. A boiled solution that consisted of 1% w/v agarose solution was used to seal the equilibrated IPG strip onto the top of the resolving gel. 2DGE was performed in 25 L of Tris-glycine-SDS electrophoresis buffer that consisted of 192 mmol/L glycine, 25 mmol/L Tris-base, and 0.1 % w/v SDS at 25°C with a constant voltage .After IEF, an Ettan DALT II system  was used. An Ettan DALTsix multiple casters  was used to cast the 12% PAGE resolving gel (250 × 215 × 1.0 mm). The resolving-gel solution was made by mixing 75 ml of 1.5 mol/L Tris-HCl pH 8.8, 90 ml of 400 g/L acrylamide/bis-acrylamide , 3 ml of 10% ammonium persulfate, 150 ml deionized distilled water (ddH2 for 1 h 20 min) with an Amersham Pharmacia Biotech Nova Blot semi-dry transfer instrument. The proteins on the PVDF membrane were blocked for 1 h at room temperature with a solution (100 ml) of 0.3% bovine serum albumin/phosphate-buffered saline (BSA/PBS) with 0.2% Tween 20 and 0.1% sodium azide (PBST). The proteins on the PVDF membrane were incubated for 1 h at room temperature with rabbit anti-hPRL antibodies that were diluted (v:v = 1:1000) in a 0.3% BSA/PBST solution. After incubation with the primary antibody, the PVDF membrane was washed with 200 ml PBST solution (15 min × 4) and rinsed twice with ddH2O. The secondary antibody, goat anti-rabbit alkaline phosphtase-conjugated IgG was diluted (v:v = 1:4000) in a 0.3% BSA/PBST solution. The solution was added to the blots for 1 h at room temperature. The membrane was washed with 200 ml PBST solution (15 min × 4), and proteins were visualized with 5-bromo-4-chloro-3-indolyl phosphate.After electrophoresis, the 2D gel was removed, and proteins in the 2D gel were transferred to a polyvinylidene fluoride (PVDF) membrane  was used to obtain accurate masses. Blank-gel experiments were conducted simultaneously to remove masses from known contaminants , matrix, trypsin, and other unknown contaminants. Each protein was identified by using the data obtained from MALDI-TOF MS PMF by searching the Swiss-Prot database 091215  with PeptIdent software.MALDI-TOF MS was used to analyze each protein that was digested with trypsin. Experiments were performed on a Perseptive Biosystems MALDI-TOF Volyager DE-RP MS . The parameters of the instrument were described . The proDeca mass spectrometer . The instrument parameters were: electron multiplier-900 V, ESI voltage 2.0 kV, and capillary probe temperature 110°C. The detailed experimental steps have been described . Tryptic peptides were analyzed with MALDI-TOF-TOF MS that operated in the reflective mode at an acceleration voltage of 25 kV over m/z 800-4000. Precursor ions close to the theoretical m/z were selected for TOF-TOF [UltraFlex III MALDI-TOF-TOF ] MS analysis. After automatic analysis, any remaining unidentified ions were manually analyzed. In the results obtained from MS, data compared to the theoretical values from the database were used to determine whether these peptides had undergone PTMs.Proteins in the 2D gel-spots that corresponded to the positive Western blot were excised and digested in the gel with trypsin. For Perspective Biosystems MALDI-TOF-TOF MS analysis, the tryptic peptide extraction was eluted directly from a liquid chromatography microcolumn onto a MALDI plate with a matrix that contained 3 mg/ml saturated α-cyano-4-hydroxycinnamic acid solution. The purified tryptic peptide mixture was analyzed (http://www.cbs.dtu.dk/services/NetPhos) (http://www.cbs.dtu.dk/services/NetNGlyc) (http://www.cbs.dtu.dk/services/NetOGlyc) (NetPhos 3.1 Server (NetPhos) , 23, NetetNGlyc) , and NetetOGlyc)  were usep = 0.05.The Chi-square test included in SPSS 22 software was used to analyze the difference in proportional ratio of PRL variants among five subtypes of pituitary adenomas, with a significance level of pI-Mr pattern, including hPRL variants v1 , v2 , v3 , v4 , v5 , and v6 .Approximately 1,200 protein spots were detected in each silver-stained 2D gel. Six protein spots were found to contain PRL (Swiss-Prot No. P01236)  in the Swiss-Prot human database. The MS/MS spectrum of the tryptic peptide 118SWNEPLYHLVTEVR131 contained a robust product-ion series that included b8, b9, b12, b13, y1, y2, y5, y6, y7, y10, y11, and y12 ions  analyzed here with the Chi-square test performed with SPSS 22 software (p < 0.05) , except for no significant difference between subtypes FSH+/LH+ and PRL+, and between subtypes FSH+ and PRL+.In the non-hormone expressed nonfunctional pituitary adenoma (NF-NFPA) group relative to controls Table , hPRL wa) Figure . The Chi72YTHGRGFITK81 with a [M+H]+ ion at m/z 1179.7 and 118SWNEPLYHLVTEVR131 with a [M+H]+ ion at m/z 1743.0. Two tryptic peptides that is the same as spot v1 were also identified in spot v4. Compared to spots v1 and v4, one more tryptic peptide 171ENEIYPVWSGLPSLQMADEESR192 with a [M+H]+ ion at m/z 2550.6 was identified in spot v6. The tryptic peptide 118SWNEPLYHLVTEVR131 of PRL in spot v6 was also analyzed with MALDI-TOF-TOF MS/MS. Those two tryptic peptides 118SWNEPLYHLVTEVR131 and 171ENEIYPVWSGLPSLQMADEESR192, were also identified at spot v5, with [M+H]+ ions at m/z 1743.0 and 2550.2, respectively.2DGE-based Western blot coupled with anti-hPRL antibody and MS was an effective method to validate hPRL variants in human pituitaries. Four hPRL variants in human pituitary, including variants v1, v4, v5, and v6, were detected with an hPRL Western blot-immunopositive reaction  and v3  were not confirmed; that finding might be due to several factors: (i) the PRL antibody was a general antibody, not specific to a given variant, and (ii) the Mr of hPRL in the spots v2 and v3 was larger than the Mr (25.9 kDa) of hPRL, obviously some adducts were added to the hPRL, which could be result in its non-reaction with the hPRL antibody. Whether the hPRL was detected with an immunoblot or not the hPRL in spots v2 and v3 was unequivocally identified with MS and MS/MS  was identified  were not detected because they were out of the range of MS acquisition , but not from mature hPRL (position 29–227).PRL in human Swiss-Prot database contains 227 amino acids position 1–227) with a signal peptide (position 1-28)  residue to aspartate (D) and of glutamine (Q) residue to glutamate (E) is an effect of protein aging, and is often observed in 2-D gels  contained in each hPRL variant in human pituitaries, six identified hPRL variants were not from splicing and truncation. Thus, PTMs might be the main factor to result in six hPRL variants. In the 2DGE map of hPRL variants . This phenomenon might arise from some PTMs with a relatively larger chemical group, such as glycosylation or phosphorylation. Glycosylation means that the protein contains one or more covalently linked carbohydrates of various types from monosaccharides to branched polysaccharides. Phosphorylation means that the protein is modified by the attachment of either a single phosphate group, or of a complex molecule such as 5'-phospho-DNA through a phosphate group at the targeted serine, threonine, or tyrosine residues. The annotation page of hPRL in the UniProt database (https://www.uniprot.org/uniprot/P01236); P01236, or PRL_HUMAN) clearly demonstrates the hPRL is a glycoprotein or phosphoprotein. Moreover, multiple phosphorylated sites at the serine (S), threonine (T), and tyrosine (Y) residues were significantly predicted by NetPhos 3.1 Server, including 14 pS sites, 5 pT sites, and 3 pY sites in the hPRL  with NetPhos 3.1 Server with a score more than 0.5.Table 7Prediction of N-glycosylation sites in hPRL (position 1–227) with NetNGlyc 1.0 Server with score more than 0.5.PRL plays an extremely important role in different vertebrate species and organ systems, such as growth, metabolism, development, reproduction, immunoregulation , behavioMoreover, PRL is involved in some endocrine-related cancers. PRL plays a vital role in the regulation of the immune responses; it interferes with inhibiting apoptosis, induces B-cell tolerance, increases antibody secretion, enhances antigen presentation, and upregulates cytokine production . PRL haspI and Mr. Therefore, the present study still used the six hPRL variants to study their differential expression profile among different subtypes of pituitary adenomas.The pituitary proteome was separated with 2DGE, followed by MS identification. Six 2D gel spots were found to contain hPRL with MS-identification, which represents six hPRL variants in human pituitaries. Of them, four hPRL variants  were confirmed with 2DGE-based Western blotting in combination with anti-hPRL antibodies and followed by MS identification. However, two hPRL variants (v2 and v3) was not detected with 2DGE-based Western blotting in combination with anti-hPRL antibodies, which is due to the factors: (i) anti-hPRL antibody was not a variant-specific antibody, and (ii) those two hPRL variants might have different properties including different PTMs and unknown factors to cause its no-reaction with anti-hPRL antibody. Anyway, this present study directly MS-identified the hPRL in six different 2D gel spot with different There are many factors that might produce protein variants: (i) Estimates of DNA level variation. Major sources of human protein variation include the encoding of single nucleotide polymorphisms and mutations. (ii) The main source of RNA level variation. Alternative splicing is a key factor in transcriptome complexity and regulating complex human characteristics. (iii) Errors in translation. Errors in protein translation provide a very large potential source of proteome expansion, especially in stressed or aging cells. (iv) PTMs. Due to PTMs, the potential number of proteoforms can increase exponentially . PTMs miMr but obviously different pI, which might be mainly due to deamidaton. The hPRL variants v2 and v3 with a slightly larger Mr relative to hPRL (position 1–227) might be mainly due to the glycosylation and phosphorylation. Therefore, PTMs might be the main reason to produce hPRL variants. Furthermore, we confirmed that the amino acid sequence of six identified hPRL variants in human pituitaries contained the signal peptide (position 1–28), which is the hPRL prohormone (position 1–227), but not the mature PRL (position 29–227).The present study found that six PRL variants that are derived from the same PRL gene in human pituitaries were not due to alternative splicing, but rather to PTMs, including deamidation, glycosylation, and phosphorylation. The hPRL variants v1, v4, v5, and v6 on the 2DGE map had a very similar The study of the hPRL signaling pathway network is helpful to understand its functions; explore molecular mechanism of PRL-related tumors such as pituitary adenomas, including prolactinomas; develop effective therapeutic drugs; and discover tumor biomarkers . Only afThe present study clearly demonstrated the existence of six hPRL variants in human pituitaries with 2DGE and MS analyses, and its differential expression profile of six hPRL variants among different subtypes of pituitary adenomas; and confirmed that those six hPRL variants in human pituitary tissues were derived from hPRL prohormone with 227 amino acids (position 1–227), but not from mature hPRL with 199 amino acids (position 29–227).However, mature PRL is secreted by prolactotroph cells concentrated on both sides of the posterior pituitary gland and enters the bloodstream after secretion. After blood transport, PRL will bind to its specific receptor before it can play a corresponding physiological role. PRL in pituitary tissue is difficult to detect with routine examinations, but PRL in blood is relatively easy to detect. Under normal circumstances, the level of PRL in the blood plays its role in a certain range. The abnormal secretion of PRL is related to many diseases, such as hyperprolactinemiaemia, tumor, and autoimmune diseases. Studies have shown that PRL has a direct or indirect relationship with many diseases. Whether these diseases are associated with serum PRL variants remains unknown. Therefore, it is necessary in our future studies to in-depth investigate the mechanisms and function of different PRL variants and the status of serum PRL variants. The present study provides the basis for us to in-depth study serum PRL variants and their functions.−, FSH+-, LH+-, FSH+/LH+-, and PRL+-adenomas). Moreover, six hPRL variants in human pituitaries derived from hPRL prohormone (position 1–227) with different PTMs such as deamidation, glycosylation, and phosphorylation, but not from mature hPRL (position 29–227). Those findings provide novel insights into the functions, and mechanisms of action, of hPRL in human pituitary and in PRL-related diseases, and into the potential clinical value in pituitary adenomas. Furthermore, the hPRL variants involved signaling pathways that might clarify the biological functions of different PRL variants and their potential clinical significance, and contribute to the development of drugs that block the PRL signaling pathway for clinical treatment.2DGE, 2DGE-based Western blot coupled with anti-hPRL antibody, MS, and bioinformatics were used to identify hPRL variants in human pituitaries, and their differential expression profiles among different pituitary adenomas. Six hPRL variants in human pituitaries were identified, including two hPRL variants that were not detected by Western blotting. The differential expression patterns of six hPRL variants were significantly different among five subtypes of pituitary adenomas (NFSQ analyzed data, prepared figures and tables, designed, and wrote the manuscript. YY collected tissue samples, performed clinical explanation, and carried out bioinformatics analysis and partial revision of manuscript. NL, TC, XW, and JL participated in experiments and partial data analysis. XL collected tissue samples and performed clinical diagnosis. DD participated in design, instructed, and critically revised manuscript. XZ conceived the concept, designed experiments, and manuscript, instructed experiments and data analysis, supervised results, coordinated, critically revised/wrote manuscript, and was responsible for its financial supports and the corresponding works. All authors approved the final manuscript.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.


Roots are important plant ground organs, which absorb water and nutrients to control plant growth and development. Phytohormones have been known to play a crucial role in the regulation of root growth, such as auxin and ethylene, which are central regulators of this process. Recent findings have revealed that root development and elongation regulated by ethylene are auxin dependent through alterations of auxin biosynthesis, transport and signaling. In this review, we focus on the recent advances in the study of auxin and auxin–ethylene crosstalk in plant root development, demonstrating that auxin and ethylene act synergistically to control primary root and root hair growth, but function antagonistically in lateral root formation. Moreover, ethylene modulates auxin biosynthesis, transport and signaling to fine-tune root growth and development. Thus, this review steps up the understanding of the regulation of auxin and ethylene in root growth. Oryza sativa) and maize (Zea mays), have fibrous root systems composed of a primary root, lateral roots and crown roots  , an inhibitor of YUC activity, suppresses ethylene-inhibited root growth [OsEIN2 and OsEIL1 overexpression transgenic lines and OsEIL1 directly activates the expression of OsYUC8 to modulate auxin biosynthesis and ethylene-inhibited primary root elongation [The auxin–ethylene crosstalk during root growth has been proposed based on several studies ,37. In AA levels ,51,64. IA levels ,51. By sEI8/TAA1 ,64. WEI2 effects . The sam pathway . These m pathway ,66,67,68UC genes ,66. EIN3in roots . Using yt growth ,70,71. Rongation .Arabidopsis, mutants of auxin receptor tir1 and auxin response AUXIN RESISTANT axr2-1 and axr3-1, mutants in IAA7 and IAA17, respectively, were shown an ethylene-resistance root phenotype [In addition to auxin biosynthesis mutants, mutants related to auxin transport, perception and signaling also display abnormal responses to ethylene ,51,64,72henotype ,51. In rhenotype . In concLateral roots are an essential component of root system architecture that maximizes the ability of the root system to acquire nutrients, therefore enabling the plant to adapt readily to the changes of the environment cues ,74. LateArabidopsis roots, gain-of-function yuc mutant (yuc1D), superroot1 (sur1) and sur2 showed an increase in endogenous auxin levels and lateral root number [auxin resistant 1 (aux1), like aux1 3 (lax3), pin-formed (pin) multiple mutants, gnom and auxin resistant 4 (axr4), have reduced lateral root number or disturbed lateral root primodium [osaux1 mutant has a reduced number of lateral roots, accompanied by decreased endogenous auxin levels [Arabidopsis gain-of-function Aux/IAA mutants, such as iaa1, iaa3, iaa14/solitaryroot 1 (slr1), iaa18, iaa19/massugu 2 (msg2), iaa28 [osiaa11, osiaa13 and osiaa23 from rice, showed aberrant lateral roots, reduced lateral root number, or even complete absence of lateral root formation [arf7 arf19 double mutant, showed complete absence of lateral root formation [It is well known that the phytohormone auxin is involved in every stage of lateral root formation ,77. Endot number ,82. The n levels ,83,84. Fn levels ,19, whicn levels . A serie), iaa28  and osiaormation ,88,89. AArabidopsis and tomato [eto1 mutant) or signaling (in ctr1 mutant) reduced lateral root formation [etr1 and ein2), form an increased number of lateral roots [Ethylene also plays an important role during the initiation and growth of lateral roots. For reviews of ethylene effects on lateral roots development, we refer to . Treatmed tomato ,92. Simiormation . By contal roots , suggestaux1, lax3, pin3 and pin7, were less sensitive to the inhibition of lateral root formation and stimulation of auxin transport following treatment with ACC [pin2 and abcb19 mutants exhibited normal responses to ACC [Arabidopsis seedlings show a local depletion of PIN3 and PIN7 abundance in the region just below developing lateral root primordia, resulting in a local auxin accumulation at the lateral root primordia, thus promoting the formation of lateral roots. ACC treatment increased the abundance of transcripts PIN3 and PIN7, thus increasing rootward auxin transport, which suppresses the local auxin maxima at lateral root primordia, inhibiting their outgrowth [Auxin and ethylene have antagonistic effects in lateral root development. Ethylene reduces DR5erv:GFP expression in the regions where lateral roots emerge, suggesting a locally reduced auxin responsiveness . Mutantswith ACC ,93. By cutgrowth .Adventitious root, also known as crown root, constituting the major part of the monocotyledonous root system, emerges from any non-root tissue and is produced both during normal development and in response to stress conditions, such as flooding, nutrient deprivation and wounding during the growing period .YUCCA genes in rice cause massive proliferation of crown roots. On the other hand, disruption of TAA1 greatly reduces crown root development [CROWNLESS ROOT 1 (CRL1)/ADVENTITIOUS ROOT LESS 1 (ARL1) encodes an ASYMMETRIC LEAVES 2 (AS2)/LATERAL ORGAN BOUNDARIES (LOB) transcription factor, which is transcriptionally regulated by the AUXIN RESPONSE FACTOR 16 (ARF16), acts as a positive regulator for crown root formation [crl5 mutant produced fewer crown roots and displayed impaired initiation of crown root primordia. Its expression can also be induced by exogenous auxin treatment and may be a direct target of an ARF [OsIAA genes were down-regulated in crl6, linking CRL6 to auxin regulatory network [WUSCHEL-related Homeobox (WOX) gene, WOX11, is involved in the activation of crown root emergence and growth. Its expression is induced by auxin and functions downstream IPyA pathway [Similar to other development processes, adventitious root formation is highly dependent on auxin action . Overexpelopment ,49. Manyormation ,97. CRL4ormation . The crlf an ARF . CRL6 in network . The WUS pathway ,102,103.Ethylene is another important phytohormone in root development through interacting with auxin. However, ethylene function in crown root development remains unknown. In rice, OsERF3, which encodes an AP2/ETHYLENE-RESPONSIVE FACTOR (ERF) protein, negatively regulates ethylene biosynthesis and act as a WOX11-interacting partner involved in rice crown root development . Moreovesur1 and yuc1D mutants) promoted root hair elongation [ein2-1 mutant and mutant aux1 on an eto1 background suppressed the long-root-hair phenotype of eto1 mutant [ROOT HAIR DEFECTIVE 6-LIKE 4 (RSL4) promoter and directly activate its expression [RSL4 to promote root hair elongation [Plant root hairs are unicellular extensions of root epidermal cells that increase the root surface to enhance water and nutrient uptake and improve soil anchorage. Multiple phytohormones, including auxin and ethylene, play vital roles in regulating cell development in the root epidermis ,106. Conongation ,46. Moreongation . These d1 mutant . Recent pression , thus prpression . RSL4 alongation . HoweverArabidopsis root development has been elucidated. However, their interaction in other plants is still unclear. In this review, we summarize the recent advances in the study of auxin–ethylene crosstalk in plant root development (Over the years, the crosstalk between ethylene and auxin in elopment , step upPlant root systems adapt rapidly to environmental changes and enable the plant to survive adverse conditions. In response to environmental cues, root growth is adapted through the modulation of endogenous auxin levels . HoweverArabidopsis, such as EIN3, ERF1 and PIF4 [Ethylene and auxin are known to interact in the regulation of root growth. Several factors function as crosstalk nodes between ethylene and auxin have been identified in and PIF4 ,66,67,68


Polyamines not only play roles in plant growth and development, but also adapt to environmental stresses. Polyamines can be oxidized by copper-containing diamine oxidases (CuAOs) and flavin-containing polyamine oxidases (PAOs). Two types of PAOs exist in the plant kingdom; one type catalyzes the back conversion (BC-type) pathway and the other catalyzes the terminal catabolism (TC-type) pathway. The catabolic features and biological functions of plant PAOs have been investigated in various plants in the past years. In this review, we focus on the advance of PAO studies in rice, Arabidopsis, and tomato, and other plant species. Polyamines (PAs) are aliphatic amines of small molecular mass that are involved in various biological processes ,2. The pThe homeostasis of cellular PA levels, being well regulated by a dynamic balance of biosynthesis and catabolism, is most important for maintaining normal growth and development in plants. The PA biosynthetic pathway has been well elucidated ,34,35, hN-carbamoylputrescine amidohydrolase . Besides, some plants have the ornithine decarboxylase  which catalyzes ornithine to Put directly [ODC genes. Secondly, the diamine Put is converted to triamine Spd by Spd synthase . Finally, Spd is further converted to Spm or T-Spm, two tetraamine isomers, by Spm synthase  and T-Spm synthase , respectively [S-adenosylmethionine (dcSAM) produced from methionine in two sequential reactions catalyzed by methionine adenosyltransferase and S-adenosylmethionine decarboxylase (SAMDC), respectively. These aminopropyl groups participate in the biochemical reaction of Spd, Spm, and T-Spm biosynthesis processes. Additionally, norspermidine (NorSpd) and norspermine (NorSpm), having been found as “uncommon PAs” due to their limited distribution in nature, are predicted to be synthesized either successively by each specific aminopropyl transferase (APT) or by a single APT with broad substrate specificity from 1,3-diaminopropane  [Plant PA biosynthesis is rather short, which starts mainly from arginine (Arg). The pathway is briefly shown in directly , but Araectively ,19,47,581,3-DAP) .N-(3-aminopropyl)-4-aminobutanal, respectively, as well as hydrogen peroxide (H2O2) which acts as an important signaling to regulate the expression of numerous genes relative to the stress response in the back conversion (BC-type) pathway; in addition to 1,3-diaminopropane and H2O2 in the terminal catabolism (TC-type) pathway [PA biosynthetic pathways have been well investigated. In contrast, the knowledge on PA catabolism in plants is still fragmental though scholars reported some new findings in the past years. Two kinds of enzymes are involved in PA catabolism. Namely, one is a copper-dependent diamine oxidase  and the other is a flavin adenine dinucleotide (FAD)-dependent polyamine oxidase . PAOs, using FAD as cofactor, catalyze Spd and Spm to produce 4-aminobutanal and  pathway ,53,54,60Vitis vinifera PAO (VvPAO6). Currently, almost all PAOs of the rice and Arabidopsis have been well determined, and we recently identified the tomato PAOs. Thus, we will review on the advance of PAOs from these three species, as well as other plant species, in this manuscript.Up to now, more and more plant PAOs have been cloned and functionally identified. In PAOs exist in rice, orderly named as OsPAO1~OsPAO7 [OsPAO3~5 are similarly and highly expressed in two-week-old seedlings and mature plants, whereas the other four OsPAO members are only expressed at very low levels in all tissues. Especially, OsPAO2, OsPAO6, and OsPAO7 are expressed at almost negligible levels, as shown in Atpao5-2, the transgenic plants restored normal T-Spm sensitivity, which can grow in the presence of low levels of T-Spm; whereas the control with the introduction of OsPAO3—a peroxisome localized PAO—into Atpao5-2 mutants did not complement the phenotype [OsPAO1 and AtPAO5 are functionally orthologous genes in Arabidopsis and rice [Ono et al. reported that seven 1~OsPAO7 . He and henotype . These gand rice .pro:GFPOsPAO7 transgenic rice plants showed that OsPAO7 is specifically expressed in anther walls and pollens with an expressional peak at the bicellular pollen stages, as shown in OsPAO7 might have special roles in floral differentiation, especially in anther development and fertility, as shown in OsPAO6 obtained from the National Center for Biotechnology Information (NCBI) public database is incorrect [OsPAO6, encoding a 497-amino acid protein, shows 92% identity and very similar protein tertiary structures to OsPAO7, and it is subcellularly localized to the plasma membrane, suggesting that OsPAO6 possibly also acts like OsPAO7 having the TC-type activity [OsPAO6 was induced by exogenous jasmonic acid, implying OsPAO6 may be involved in stress tolerance [OsPAO2 derived from NCBI might be incorrect like the case of OsPAO7.Interestingly, our group found that OsPAO7, with high amino acid identity and very similarly predicted protein 3-D structures to ZmPAO1, which is the best characterized maize PAO catalyzed TC-type reaction, is subcellularly localized to the apoplastic space with the aid of a signal peptides  and transmembrane domains  in its N-terminal, as shown in ncorrect . They suncorrect . The coractivity ,49,60. Folerance ,49,60. HOsPAOs remains limited. Chen et al. found that OsPAO1~7 is most important for rice germination compared to the subfamilies’ members OsPAO8~11 encoding histone lysine-specific demethylases, especially OsPAO5 which probably regulates rice seed germination via PAO-generated H2O2 signaling to mediate coleorhiza-limited rice seed germination [Up to now, the knowledge of the biological functions of mination .OsPAOs in various developmental and physiological processes, molecular and genetic approaches like CRISPR/Cas9-mediated loss-of-function mutants and ubiquitin promoter enhanced overexpression transgenic plants should be generated.Above all, two different kinds of PAOs exist in rice; one is BC-type , the other is TC-type , as shown in PAOs, named as AtPAO1 to AtPAO5. The recombinant protein of the former four AtPAOs, AtPAO1~4, have been homogenously purified and characterized [The Arabidopsis genome contains five cterized ,62,64,65cterized ,51. In dcterized ; AtPAO2~cterized ,61,62,64PAOs showed different expression patterns. AtPAO1 is specifically expressed in the root transition region (between the meristematic and elongation zones of the root) and anther tapetum [AtPAO1 is specifically expressed in anthers [AtPAO1 was reported to be involved in environment stress tolerance [AtPAO1 may also play roles in root development and fertility, as shown in AtPAO2 is mainly expressed in the root and shoot meristematic area, the vein of rosette leaves, as well as the anthers, suggesting that AtPAO2 might function in the development of roots, shoots, leaves, and flowers, as shown in AtPAO3 and AtPAO4 display similar expression patterns, which are expressed in all tissues and whole growth stages, especially in roots, leaves, and flowers, suggesting that these two members may mediate various significant growth processes, as shown in pao4-1 and pao4-2, two independent lines of AtPAO4 loss-of-function mutants, have 10-fold higher Spm levels compared to wild type, and delay dark-triggered senescence [AtPAO5, is expressed in all developmental stages, with strongest expression in roots, stems, leaves, and floral organs, as shown in Five Arabidopsis  tapetum , and Tak anthers . AtPAO1 olerance ,65, and nescence . The lasAtPAO5 is a relatively completely explained Arabidopsis PAO, and its gene product AtPAO5 has been successfully characterized and its biological function also has been explored [AtPAO5 regulates stem elongation and the rosette leaves’ development, as shown in AtPAO5 T-DNA insertion mutants, pao5-1 and pao5-2, both of which show about 2-fold higher levels of T-Spm, still maintain normal levels of Put, Spd, and Spm compared to the wild type controls [pao5-1 and pao5-2 mutants exhibit more rosette leaves, and shorter and fewer inflorescence stems at the two-month-old stage. Further genetic and morphology analysis suggested that AtPAO5 plays roles in Arabidopsis growth and development through oxidizing T-Spm [atpao5-2 and atpao5-3, two independent loss-of-function mutants of AtPAO5, show higher T-Spm contents, mediate metabolic and transcriptional reprogramming, and enhance salt-related stress tolerance [AtPAO5 also plays roles in the control of proper xylem differentiation through interplaying between auxin and cytokinins [AtPAO5 mutant with higher T-Spm levels shows the similar phenotypes as acl5 (tkv) and bud2 mutants, which only contain very low or even zero T-Spm content [explored ,67,68. Aexplored . Our forcontrols . The paong T-Spm ,51. Ahoung T-Spm . atpao5- content ,62,72,73AtPAO1~4) biological functions need to be further unveiled in the future.Taken together, all five Arabidopsis PAOs catalyze BC-type reactions and mediate  the entire developmental processes in plants, as shown in S. lycopersicum cv. Chiou, the expression of PAO peaked at ImG1 (fruits 0.5 cm in diameter) and ImG2 (fruits 1 cm in diameter) stages, suggesting PAO participates in developmental processes of the fruits, including the cell wall maturation [ZmPAO (S-ZmPAO) transgenic tomato plants have slightly larger leaf sizes and higher antioxidant enzyme activities; in contrast, the antisense-ZmPAO (AS-ZmPAO) transgenic tomato plants contain lower chlorophyll content index, smaller leaves, and less biomass, as well as an increment in Ca2+ when responding to salt stress [S-ZmPAO and AS-ZmPAO transgenic plants suggested that apoplastic PAO play important roles in plant growth and stress responses [Solanum lycopersicum) has seven PAO genes in its genome, which were orderly named as SlPAO1 to SlPAO7 [SlPAO2~5, sharing high identity (over 64%) of amino acid and showing quite similar genome organization and predicted tertiary structures, have similar tissue expression patterns [SlPAO2~4 are ubiquitously and highly expressed in the whole growth processes and all tissues, predominantly in anther, Br (breaker stage fruit), and Br+2 (two days post breaker stage fruit) [SlPAO2~4 may play dominant roles in all stages of growth especially in floral development and fruit maturity in tomato, as shown in SlPAO1 is expressed relatively lower than SlPAO2~4 in all of the vegetative tissues and anthers [SlPAO6~7, sharing quite similar identity of amino acid and very similar intron-exon organization and protein 3-D structures, are lowly expressed in vegetative and reproductive tissues, but had relatively higher expression in roots, stems, buds, and anthers than in the fruit [PAOs may mainly function in vegetative and anthesis tissues but not in fruit, as shown in SlPAOs respond to abiotic stresses , oxygen species (H2O2 and methylviologen), phytohormones , as well as PAs , implying that tomato PAOs possibly have various functions in stress tolerances, as shown in SlPAOs possibly play vital roles in different tissues and developmental stages, especially in floral development and fruit repining. To better explain the mechanism of polyamine catabolism and biological roles of SlPAOs, more biochemical and genetic experiments are required.Transgenic tomato plants overexpressing maize PAO (MPAO) exhibit tissue damage with lower chlorophyll content, lower photochemical efficiency of photosystem II (PSII), and DNA fragmentation compared to wild type, suggesting that the increased PAO activity cannot cope with the reactive oxygen species (ROS) generated by environmental factors . In S. lturation . Gémes et stress . The pheesponses . Most reo SlPAO7 . SlPAO2~patterns . Besidese fruit) , suggest anthers . What ishe fruit , suggest2O2 signaling which is generated by stress-induced PAO activity leading to Spd, Spm, and T-Spm oxidation [Botrytis cinerea infection, suggesting that PA back-conversion and/or terminal catabolism were involved in PA homeostasis under stress conditions [Medicago falcate [salinity tolerance 1 (st1), a wheat salinity-tolerant line, the expression of PAO genes showed high expression levels, suggesting that PAO genes may have important functions in salinity tolerance [Recently, besides these three model plants , some other plant species have also been studied on PAO catabolism, and PAO biological functions. Plant PAOs play important roles in various stress tolerance and the programmed cell death (PCD) events through mediating Hxidation ,96,97,98 falcate ,76, sugg falcate ,100,101.olerance .Selaginella lepidophylla back-converts Spm and T-Spm to Spd and Nor-Spd, respectively [Atpao5, with the reduction of T-Spm content to almost normal levels of wild type, which strengthens the claim that T-Spm homeostasis is required for plant development and growth [Citrus sinensis); their results indicated that six PAO genes (CsPAO1–CsPAO6) exist in sweet orange, and they also found that CsPAO3 may have potential roles in PA back conversion in plants, while CsPAO4 catalyzes Spd and Spm as substrates for terminal catabolism [CsPAO4 showed growth inhibition under salt stress caused by the elevation of H2O2 which leads to oxidative damages [MdPAO2 was obviously upregulated in apple fruit by elevating the CO2 concentrations under low-temperature/low-O2 storage for up to sixteen weeks, suggesting that MdPAO2 is involved in respiratory activities in apple fruit storage under multiple abiotic stresses [Brachypodium distachyon, and they found that BdPAO2 and BdPAO4 possibly are localized to peroxisomes [Previously, Sagor and his colleagues reported that SelPAO5 from ectively . It is dectively . Most red growth . Besidestabolism ,71. The  damages . What isstresses . Furtheroxisomes . Additiooxisomes .2O2 under Al toxicity; in contrast, the CW-PAO activity was markedly inhibited by Put application, and subsequently reduced H2O2 accumulation in roots under Al stress, suggesting that Put plays an important protective role against Al-induced oxidative stress via inhibiting the PAO activity with lower H2O2 production [Plant PAOs play significant roles in metal toxicity tolerance. Aluminum (Al), copper (Cu), and cadmium (Cd), etc. are phytotoxic to plants at high concentrations ,105,106.oduction . Similar2O2 [2O2 for cell-wall strengthening and lignosuberization events, and the peroxidase substrate is provided in these cells throughout AZ development [2O2 to generate ·O2- through enhanced substrate availability and subsequently maintain maize leaf elongation under saline stress [2O2 which is required by vascular differentiation and the process of polymerization of lignin precursors into lignin [Atpao3, a loss-of-function mutant of AtPAO3 which oxidizes Spd in peroxisomes [2+ currents [Plant PAOs have important roles in plant growth and development. Around fifteen years ago, the functions of the maize PAO were investigated by the Rea group and the Cona group separately, and they found that the maize PAO plays roles in cell-wall maturation and root differentiation by producing H2O2 ,108. Gomelopment . Moreovee stress . What iso lignin . Atpao3,oxisomes , shows rcurrents . Furthercurrents .AtPAO2~4 were speculated to be localized to peroxisomes [OsPAO3~5 are situated in peroxisomes [BdPAO2 and BdPAO4 from Brachypodium distachyon [BrPAO2~4 from Brassica rapa [CsPAO2~3 from Citrus sinensis [SlPAO2~4 from tomato [MdPAO2~4 were predicted to localize in peroxisomes, whereas MdPAO1 and MdPAO5~6 were predicted to be cytosolic proteins [Arabidopsis have two different localizations; the AtCuAO2 and AtCuAO3 are localized to peroxisomes, while the AtAO1 and AtCuAO1 are localized to apoplast [In Arabidopsis, oxisomes ,62,64,65oxisomes ,47. Besistachyon , BrPAO2~ica rapa , CsPAO2~sinensis , and SlPm tomato  were prem tomato ,81. In tm tomato . The MdPproteins . In addiapoplast .These peroxisomal PAOs shared high identity (over 57% compared to OsPAO3 which was set as 100%), as shown in 2O2 generation, remain fragmentary. Thereby, the significance of the functional difference between peroxisomal or cytoplasmic PAOs and apoplastic PAOs remains to be clarified and should be addressed in future work.It is suggested that the peroxisomal PAOs possibly play significant roles in plant growth processes, especially in floral development, as shown in 2O2 is generated by PAOs. Subsequently the H2O2 signal participates in the developmental processes such as root growth, xylem differentiation, pollen tube growth, fruit development, etc., as shown in 2O2 accumulation, leading to a ROS burst which may result in death, as shown in In the past years, some PAO genes were cloned and functionally identified from different plant species. Some research groups focus on the PA catabolism pathway, meanwhile, more and more researchers pay intense attention to the biological roles of PAOs. As it is known, when plants grow under normal conditions, the intracellular PAs maintain homeostasis, and the normal level of HPAOs and most rice PAOs have been identified, but their biological roles remain largely unclear. Meanwhile, the tomato PAOs have been cloned, but its catabolic activities and biological functions are still unknown. What is more, the exact roles of the highly conserved peroxisomal PAOs in plants are still fuzzy. Furthermore, why does rice have two different types of PAO catabolic pathways (the BC-type and TC-type pathway)? In addition, the exact mechanism of PA metabolism and the PA-cycle—PA exodus—as well as the possible ratio between the back-conversion and terminal catabolism in plants needs to be uncovered. Finally, what is the possible relationship between PAO and proline when plants fight against environmental stresses? To fully understand the roles of PAOs in plant development and stress interactions, intensive studies are required via generating loss-of-function mutants and overexpression transgenic plants which will greatly help further explore the biochemical and physiological roles of these PAOs.Recently, the enzyme features of all the Arabidopsis


Induced pluripotent stem cells (iPSCs) offer the unique possibility for directed differentiation into mature podocytes. The current differentiation protocol to generate iPSC-derived podocyte-like cells provides a robust and reproducible method to obtain podocyte-like cells after 10 days that can be employed in in vitro research and biomedical engineering. Previous published protocols were improved by testing varying differentiation media, growth factors, seeding densities, and time course conditions. Modifications were made to optimize and simplify the one-step differentiation procedure. In contrast to earlier protocols, adherent cells for differentiation were used, the use of fetal bovine serum (FBS) was reduced to a minimum, and thus ß-mercaptoethanol could be omitted. The plating densities of iPSC stocks as well as the seeding densities for differentiation cultures turned out to be a crucial parameter for differentiation results. Conditionally immortalized human podocytes served as reference controls. iPSC-derived podocyte-like cells showed a typical podocyte-specific morphology and distinct expression of podocyte markers synaptopodin, podocin, nephrin and WT-1 after 10 days of differentiation as assessed by immunofluorescence staining or Western blot analysis. qPCR results showed a downregulation of pluripotency markers Oct4 and Sox-2 and a 9-fold upregulation of the podocyte marker synaptopodin during the time course of differentiation. Cultured podocytes exhibited endocytotic uptake of albumin. In toxicological assays, matured podocytes clearly responded to doxorubicin (Adriamycin™) with morphological alterations and a reduction in cell viability after 48 h of incubation.Podocytes play a critical role in glomerular barrier function, both in health and disease. However,  Podocytes, also known as visceral epithelial cells, play a key role in the glomerular filtration barrier and the maintenance of glomerular function ,2. PodocPodocytes are highly specialized, terminally differentiated cells playing a pivotal role in maintaining the glomerular filtration barrier and producing growth factors for surrounding cells, namely mesangial and endothelial cells ,7. They in vivo, making them vulnerable to various insults leading to glomerular sclerosis [in vitro, making it difficult to obtain adequate quantities of podocytes at a differentiated stage for basic research [In their mature, fully differentiated state, podocytes have a limited capacity to proliferate clerosis . The limresearch –13. Due research ,15. The research . Howeverresearch –19.in vitro podocyte research [A conditionally immortalized human podocyte cell line has been established by transfection of podocyte primary cells with a temperature-sensitive SV40 large-T antigen gene construct . Howeverresearch –33.In this respect, the directed differentiation of human iPSCs into glomerular podocytes was described in two recent studies ,35. Songhttp://stembancc.org) [The aim of the current study was to evaluate the reproducibility and robustness of currently available podocyte differentiation protocols and to optimize the protocols accordingly. Here, we report on a direct differentiation of human iPSCs into functional podocytes, based on the protocols of Ciampi et al.  and Songncc.org) . iPSC-dencc.org) ,35 diffeThe differentiation protocol developed in the present study is based on a direct path to mature podocyte-like cells without intermediate progenitor stages, using a combination of BMP7, activin A and retinoic acid. In modification of earlier protocols ,35, the 2. After 4 days in culture, iPSCs were detached by Accutase to yield single cell suspensions to start differentiation into podocyte-like cells. Cells were counted and 9,000, 15,000 and 40,000 cells/cm2 were then plated on Geltrex-coated multi-well plates for differentiation experiments. The results of the initial plating experiments are shown in 2 for the undifferentiated iPSC pre-cultures, and after 4 days in culture, 9000 cells/cm2 for efficient differentiation into podocyte-like cells, respectively. The elaborated plating protocol ensured optimal growth conditions for a maximum of evenly distributed cells at appropriate density for cell enlargement and development of podocyte-like structures, respectively. It also ensured sufficient cell numbers and biomass to be obtained for down-stream experiments. These seeding densities were further critical for the reproducibility and robustness of the protocol, as higher seeding densities of undifferentiated iPSCs at 200.000 and 400.000 cells/cm2 resulted in significantly lower numbers of podocyte-like cells or no differentiation into podocyte-like cells, since cells did not attach on day 1 ; SBNEO3-01 (SBNEO); SFC018-03-0(SFC018) and SBAD2-01 (SBAD2). After detachment with Accutase, single iPS cells were gently suspended directly in differentiation medium (medium M1), containing activin A, bone morphogenetic protein (BMP7), and retinoic acid and seeded on growth factor reduced basement membrane matrix (Geltrex)-coated surface. After ten days in differentiation media, differentiated podocyte-like cells could be further maintained for at least another 10 days in maintenance medium (medium M2), which was devoid of differentiation factors activin A, BMP7, and retinoic acid  for further research.The time course of differentiation of iPSCs into podocyte-like cells was monitored by determination of mRNA expression profiles of the pluripotency markers Oct4 and Sox-2, and of synaptopodin, a podocyte-specific marker, by quantitative real-time PCR (qPCR). As depicted in The temporal changes of iPSC during differentiation towards a podocyte-like morphology were assessed by phase-contrast microscopy . Cells cimmunofluorescence and Western blot anaylsis. Synaptopodin, an actin-associated protein .In order to test for podocyte-specific functions of differentiated cells, two functional assay regimes were applied: (a) the endocytic uptake of albumin and (b) the susceptibility of podocyte-like cells to doxorubicin , a nephrotoxic anti-cancer agent is, aside its cardiotoxicity, known to be specifically toxic for glomerular podocytes. DOX leads to podocyte foot process effacement and increased glomerular permeability causing proteinuria. DOX-associated nephropathy is caused by interfering with actin reorganization of podocytes  and mitoPodocytes are key cells in the formation of the glomerular filtration barrier. In the complex filtration barrier network of the glomerulus, podocytes are the most vulnerable components. Most glomerulopathies are associated with podocyte injury causing various types of diseases, that may account for approx. 90% of end-stage kidney disease . When poin vitro cultures of human podocytes gained specific importance. However, there are intrinsic difficulties of growing differentiated podocytes in in vitro culture, that have been discussed extensively [Thus, the central role of podocytes in glomerular dysfunctions necessitates reliable model systems to study normal and impaired podocyte functions at the cellular and subcellular level. In this respect, ensively –13. MatuThe first strategy was applied using a conditionally-immortalized human podocyte cell line carrying a temperature-sensitive mutant of the SV40 large-T antigen . Therebyin vitro toxicity testing and drug safety assessment, and for tissue engineering and cell therapy applications [The second strategy is the employment of human iPSC. In 2007 Takahashi and Yamanaka discovered a technique to reprogram adult human cells into pluripotent stem cells , which eications ,29,45.in vivo stages of embryonic kidney development, with differentiation of iPSC into intermediate mesoderm and metanephric mesenchyme, and the induction of nephron progenitor cells, thereby recapitulating embryonal developmental stages of the kidney in vitro [Furthermore, human iPSC opened the possibility of directed differentiation into all cell types of the nephron . Recent in vitro ,46,47.Here we present a method to directly differentiate human podocytes from human induced pluripotent stem cells (iPSCs) in a one-step protocol, using a cocktail of retinoic acid, activin A, and BMP7. Already published protocols ,35 were Characteristic podocyte morphology, such as long spindle-like projections, fine processes, short rounded projections, and interdigitations between cells could be observed on day 10 of differentiation or earlier . The immqPCR results showed a gradual decrease in mRNA levels of pluripotency markers Oct4 and Sox-2, paralleled by an increase in podocyte marker synaptopodin mRNA during iPSC differentiation . Oct4 anhttp://stembancc.org) [in vitro at the cell level [Variations between iPSC lines are well documented. Regardless of the source of somatic cells or the method chosen, the reprogramming process itself is influenced by a number of variables that affect the reproducibility and quality of the resulting iPSCs. Each iPSC line can be considered to be a distinct clone . The repncc.org) . iPSCs fncc.org) , the genncc.org)  offers pll level ,27.2 was optimal for pre-cultures of undifferentiated iPSC. High cell numbers in dense cultures of iPSC result in rapid acidification of the mTeSR1 medium. We have recently shown that high cell densities can decrease the culture medium pH to 6.8 within 24 h. At this low pH, undifferentiated iPSC enter a transient growth arrest in G0/G1-phase [2 were proven to be most efficient for terminal differentiation  [™1 medium (StemCell Technologies). Cultures were passaged twice a week with Versene (Lonza) at a split ratio of 1:4 to 1:12.Human iPSCs used in the present study were obtained during the course of the IMI-funded StemBANCC project (ncc.org) . Human fncc.org) ,37,60 acPeriodical cell authentication of the four iPSC lines used in the present study was performed by short tandem repeat (STR) profiling  as descr™, BD Biosciences) at the permissive temperature of 33 °C in RPMI 1640 medium, supplemented with 100 U/ml penicillin, 0.1 mg/ml streptomycin, glutamine, and insulin, transferrin, sodium selenite mixture (ITS) (Sigma-Aldrich), and 10% fetal bovine serum (GIBCO) as recently described [Conditionally immortalized human podocyte-like cells, generously provided by Prof. M. Saleem, Bristol, UK  served aescribed . At 70–82. After 4 days, cultures were passaged with Accutase (GIBCO) to obtain single cell suspensions, and iPSCs were seeded on plates coated with Geltrex at a density of 9,000, 15,000 and 40,000 cells/cm2 for differentiation. In order to avoid uneven concentric distribution of cells in multiwell plates, and thus non-optimal conditions for a proper development of foot processes and interdigitations between adjacent/neighbouring cells, cells were seeded with half of the recommended volume of media per well. Back and forward and left and right gentle shaking movements were performed instead of gentle rotation before placing the plates into the incubator. The residual media was added after 5h, when cells were attached.During a series of experiments it was observed, that the initial cell densities of stock iPSCs as well as the seeding densities of the differentiation cultures play a critical role in order to obtain reproducible results in the differentiation of iPSC into podocytes. To this end, pre-cultures of undifferentiated iPSC were plated at densities of 50–100,000, 200,000 or 400,000 cells/cmIn the present one-step protocol undifferentiated iPSCs were seeded at the cell densities described above into differentiation medium containing 5 μM Rho-associated kinase (ROCK) inhibitor Y-27632  for 24 hours. Differentiation medium (medium M1) consisted of DMEM/Ham F-12 (GIBCO Cat. No. 31330–038), supplemented with 1.25% FBS (GIBCO Cat. No. 10270), 100 μM nonessential amino acids (NEAA), 15 ng/ml BMP7, 10 ng/ml activin A, and 100 nM retinoic acid , and 100 U/ml penicillin, 0.1 mg/ml streptomycin (Sigma). After 24 h, ROCK inhibitor was removed by feeding the cells with medium M1 and cultures were further maintained in M1 and fed every 2–3 days. After ten days, differentiated cells were further incubated for another 10 days in maintenance medium (medium M2), without presence of differentiation growth factors (DMEM/Ham F-12 with 2.5% FBS (GIBCO), 100 μM NEAA and 100 U/ml penicillin, 0.1 mg/ml streptomycin). Media were changed every 48 hours.Total RNA was isolated with QIAzol  from undifferentiated iPSC cultures, and from podocyte-like cell cultures at day 1, 3, 6, 9, 13 and 17 in differentiation media. 50 ng of total RNA were used for first-strand cDNA synthesis with Superscript II (Life Technologies), following the manufacturer’s instructions.™/TAMRA™ probe, cat. 4310893E, Life Technologies).Quantitative real time PCR (qPCR) was performed using TaqMan Gene Expression Master Mix (Applied Biosystems). Assays for synaptopodin , Oct4 (Hs04260367_gH) and Sox-2 (Hs01053049_s1) were purchased , based on the supplier’s validated bioinformatics pipeline for optimal alignment to the selected genes. Threshold cycle values and expression levels, respectively, were normalized against endogenous 18S RNA  or Hoechst 33342 respectively. Fluorescent images were processed using ImageJ.Cells were differentiated on glass coverslips for 10 days and fixed with 4% para-formaldehyde in PBS for 10 min at room temperature (RT). After washing three times with PBS, cells were incubated for 30 minutes in blocking buffer . Cultures were stained with primary antibodies, anti-synaptopodin (Abcam ab224491) at a dilution of 1:75, anti-NPHS2  at 1:500, anti-NPHS1  at 1:75 and WT-1  at 1:100, and incubated at RT for 30 min. The secondary antibody Alexa Fluor 568  was incubated at a dilution of 1:250 for 1 h. Either F-actin or the nucleus was counter stained using phalloidin . To assess loading variations, membranes were stripped and re-probed using a rabbit anti-tubulin antibody .Cells were harvested at different time points and homogenized in RIPA buffer (Sigma) containing a protease inhibitor cocktail (Sigma). Protein concentrations were determined using a BCA assay (PIERCE). Equal amounts of protein (15–50 μg/well) were subjected to SDS-PAGE , and transferred to PVDF membranes. After blocking in TBST with 5% non-fat milk, membranes were probed with the corresponding primary antibodies overnight at 4°C . Horseradish peroxidase-conjugated secondary antibodies, IgGs (1:5000–10.000) were incubated for 1 h at room temperature sequentially. The immunoreactive bands were visualized by a Chemiluminescence kit (ThermoFisher) and images were digitally acquired using LAS-4000 ImageQuantDifferentiated podocyte cultures were serum-starved overnight. After rinsing the monolayers with PBS, cultures were incubated with 1 mg/ml FITC-conjugated bovine serum albumin for 1 h at 4°C  and 37°C (binding and endocytosis), respectively, and for 24 h at 37°C. Albumin uptake was determined by fluorescence microscopy, using a ZEISS Axiophot Fluorescence Microscope.In doxorubicin experiments, adult, neonatal, and migraine patient-derived differentiated podocyte-like cells, and undifferentiated and differentiated human immortalized podocytes were incubated for 48 h at concentrations of 10 nM and 1 μM. The resazurin assay was used to assess metabolic activity of the cultures . ResazurAll experiments were conducted at least three times. Statistical significance was tested using the unpaired Student’s t test. P values <0.05 were considered statistically significant. See legends for more details.S1 FigiPSC were differentiated on glass cover slips, fixed and stained for synaptopodin, WT-1, podocin, and F-actin as described in methods. Red and green colours were applied post capture.(TIF)Click here for additional data file.S2 FigiPSC were differentiated on glass cover slips, fixed and stained for synaptopodin, WT-1, podocin, and F-actin as described in methods. Red and green colours were applied post capture.(TIF)Click here for additional data file.S3 FigDifferentiating, SBAD2 and SFC018 iPSC were lysed at days 0  and at days 6 and 13 and processed for Western blot analysis. Representative blots are shown.(TIF)Click here for additional data file.


Background: We investigated ion channels at the skin, including peripheral nerve endings, which serve as output machines and molecular integrators of many pruritic inputs mainly received by multiple G protein-coupled receptors (GPCRs). Methods: Based on the level of chronic kidney disease–associated pruritus (CKD-aP), subjects were divided into two groups: non-CKD-aP  and CKD-aP . Skin samples were obtained from the forearm or elbow during operations on arteriovenous fistulas. We measured ion channels expressed at the skin, including peripheral nerve endings by RT-PCR: Nav1.8, Kv1.4, Cav2.2, Cav3.2, BKCa, Anoctamin1, TRPV1, TRPA1, and ASIC. Results: Expression of Cav3.2, BKCa, and anoctamin1 was significantly elevated in patients with CKD-aP. On the other hand, expression of TRPV1 was significantly reduced in these patients. We observed no significant difference in the levels of Cav2.2 or ASIC between subjects with and without CKD-aP. TRPA1, Nav1.8, and Kv1.4 were not expressed. Conclusions: It was concluded that this greater difference in the expression of ion channels in the skin tissue including, specially cutaneous peripheral nerve endings in CKD patients with CKD-aP may increase generator potential related to itching. Itching can be caused by various stimuli, including mechanical, electrical, and chemical stimuli. Exogenous and endogenous chemical stimuli include amines, proteases, neuropeptides, inflammatory mediators, and certain drugs. However, in previous studies that compared immunostaining skin sections treated with various common pruritogens , etc.), uremic substances, and other substances causing itching , no significant difference was observed between patients with and without CKD-aP, even though the immunostaining epidermis sections were stained regardless of CKD-aP [Among uremic substances, for example, endothelin-1, which is classified of moderate molecular weight, and para-cresyl sulfate, a protein-bound uremic toxin, were reported as candidate substances that cause itching in patients with chronic kidney disease (CKD)-associated pruritus (CKD-aP) ,3. It is+ channels (Nav), Nav1.7, Nav1.8, and Nav1.9; among the voltage-gated K+ channels, Kv1.4, Kv3.4, and Kv7.2 (Kv); among the Ca2+-activated K+ channels (KCa), big conductance (BKCa); among the two-pore K+ channels, TREK1 and TRAAK; among the voltage-gated Ca+ channels (Cav), N-type (Cav2.2) and T-type (Cav3.2); among the Cl channels, calcium-activated chloride channel Anoctamin1 (TMEM16A); among the transient receptor potential (TRP) channels, TRP vanilloid 1 (TRPV1) and TRP ankyrin 1 (TRPA1); and acid-sensing ion channels (ASIC1) [Ca, Cav2.2, and Cav3.2 are expressed only in the cutaneous peripheral nerve endings, whereas TRPV1, TRPA1, Anoctamin1, BKCa, and ASIC are expressed not only in the peripheral nerve cells but also in keratinocytes at the skin.These findings suggested that itching can be caused by receptors and other signals, instead of pruritogens. Therefore, we first examined the downstream ion channels because it is difficult to measure the complex and many downstream signals from ligands (pruritogens). Expressed ion channels in cutaneous peripheral nerve endings in healthy individuals are as follows: among the voltage-gated Na (ASIC1) . Among tOur goal was to test the hypothesis that many pruritogens including uremic toxins act on ion channels on the peripheral nerve endings directly or sensitize those via GPCRs and ROS indirectly, changing the output of multiple ion channels and increase generator potential and generate action potential related to CKD-aP, resulting in encoding the amplitude, frequency and quality of impulse of the peripheral nerve. To explore the peripheral neuronal mechanisms underlying itching, we compared the expression of several ion channels at the skin, including peripheral nerve endings between CKD patients with and without CKD-aP.This cohort-sectional study was approved by the Ethics Committee of Jusendo General Hospital. . Each patient gave written informed consent. Between February 2016 and September 2018, we performed arteriovenous fistula surgery in 77 patients with CKD stage 5, either before the induction of hemodialysis (HD) or within 1 month after the induction of HD. Twenty-three patients  and one control agreed to participate in the study. The control was a patient undergoing an operation for prostatic hypertrophy, who reported no pruritus. Patients with concomitant dermatitis, e.g., atopic dermatitis or psoriasis, were excluded. The degree of pruritus was determined according to Shiratori’s Japanese classification of itching . When thWe compared age, gender, underlying disease, presence of hepatitis B or C, number of patients receiving treatment for itching, as well as levels of blood serum albumin, corrected Ca, inorganic phosphorus, intact parathyroid hormone (intact-PTH), blood serum hypersensitive C-reactive protein (hs-CRP), and blood ferritin in patients of the pruritus and non-pruritus groups.2 were obtained from the forearm or elbow immediate after commencing operations on the arteriovenous fistulae.Skin samples about 10 × 5 mm+ channels (Nav), Nav1.7, Nav1.8, and Nav1.9; among the voltage-gated K+ channels, Kv1.4, Kv3.4, and Kv7.2 (Kv); among the Ca2+-activated K+ channels (KCa), big conductance (BKCa); among the two-pore K+ channels, TREK1 and TRAAK; among the voltage-gated Ca+ channels (Cav), N-type (Cav2.2) and T-type (Cav3.2); among the Cl channels, calcium-activated chloride channel Anoctamin1 (TMEM16A); among the transient receptor potential (TRP) channels, TRP vanilloid 1 (TRPV1) and TRP ankyrin 1 (TRPA1); and acid-sensing ion channels (ASIC1) [Ca (KCNMA1), Cav2.2 (CACNA 1B), Cav3.2 (CACNA 1H), Anoctamin1 (TREM16A), TRPV1, TRPA1, and ASIC by RT-PCR. Nav1.8, Kv1.4, Cav2.2, and Cav3.2 are expressed only in the cutaneous peripheral nerve endings, whereas TREM16A, TRPV1, TRPA1, ASIC1, and BKCa are expressed not only in the peripheral nerve cells but also in keratinocytes at the skin.Expressed ion channels in cutaneous peripheral nerve endings in healthy individuals are as follows: among the voltage-gated Na (ASIC1) ,6. Among® Gene Expression Assays for human were used (TM VILOTM Naster Mix (Thermo Fisher Scientific) following the manufacturer’s protocol. cDNA was synthesized from 1 μg of total RNA. For standardization of quantitation, beta 2 microglobulin as amplified simultaneously. The expression level of each gene is presented as fold increase in the subjects showing itchy compared with control subject.Gene expression levels for ion channels were determined after reverse transcription of RNA samples by quantitative PCR by using an ABI PRISM 7000 Sequence Detector  as described previously . Pre-madere used . Total Rt-test or m × n chi-square test for categorical outcomes. p-values < 0.05 were considered to be statistically significant.All values are expressed as means ± standard deviations (SD) for normally distributed data, medians (ranges) for non-normally distributed data, and numbers (percentages). No data points were excluded. All statistical analyses were performed using the unpaired No differences were observed between the two groups in term of age, gender, underlying disease, duration of renal replacement therapy, presence of hepatitis B or C, number of patients receiving treatment for itching, or the levels of corrected Ca, inorganic phosphorus, blood serum albumin, blood serum hsCRP, and blood serum ferritin .Ca, and Anoctamin1 was significantly higher in patients with CKD-aP. On the other hand, expression of TRPV1 was significantly decreased in those with CKD-aP. No significant difference in Cav2.2 and ASIC was observed between groups. TRPA1, Nav1.8, or Kv1.4 were not expressed  (approximately 800 types in human), which then transmit itch signals to trimeric proteins, effectors, second messengers, as well as targets . These t2+ channel via GPCR [2+ channel is associated with itching in patients with atopic disease who received ACh intradermally [The Cav3.2 T-type calcium channel, which is only expressed in peripheral nerves at the skin, is associated with depolarization , action potential generation, and itching . Our stuvia GPCR ,17. For dermally . By contdermally . Furtherdermally . This indermally .2+ channels), which also is expressed in peripheral nerves, regulate the release of neurotransmitters such as glutamate and substance P in the central terminals of nociceptor neurons in the spinal dorsal horn [Ca3.2 T-channels regulate cellular excitability in the peripheral nerve endings of nociceptors, whereas Cav2.2  neurons in mice. Also, voltage-gated potassium (Kv) channels shape action potentials by controlling the repolarization phase, and they also determine the membrane potential and duration of the inter-spike interval . Based oTRPV1 are non-selective cation channels that act as biosensors for environmental and noxious stimuli, as well as changes in temperature and conditions inside the cell. In addition to capsaicin and resiniferatoxin (RTX), protons (pH < 5.7), heat (> 42 °C), and multiple other ligands (endogenous lipids and metabolic products of lipoxygenase) can directly activate TRPV1 ,26 Furth2S [2O2, in mitochondria [+ channels, as well as TRPA1 and TRPV, and also increase intracellular Ca2+ [TRPA1, which is also expressed on cells other than peripheral nerve cells can be activated by various reactive compounds, including mustard oil, cinnamaldehyde, formalin, and hydrogen peroxide, as well as noxious cold temperature, reactive oxygen species (ROS), and inflammatory lipids (4-hydroxynonenol). Wilson et al. showed that TRPA1 is the downstream target of both mas-related GPCR (Mrgpr) A3 and Mrgprc11, which act as receptors for the pruritogens chloroquine and BAM8-22, respectively . However2S . Uremic chondria . In addilar Ca2+ ,7. Thus,2+ [− from the cell [2+ via Cav3.2 T-type calcium channels.In general, membrane depolarization increases the sensitivity of Anoctamin1 to an increase in intracellular Ca2+ . Anoctamthe cell . CompareCa channels, which play a key role in controlling action potential repolarization [Ca expression, which is also expressed on cells other than peripheral nerve cells, was significantly increased in patients with CKD-aP due to the increase of intracellular Ca2+ through the Cav3.2 T-type calcium channel. If cutaneous BKCa expression reflects its expression in cutaneous peripheral nerve endings, this finding suggested that repolarization of action potentials, leading to itching, was activated; this might affect the shape or frequency of action potential impulses related to itching in cutaneous peripheral nerve ending. In fact, the frequency of itch impulses is less than that of pain impulses in peripheral c-fibers [There is evidence that members of the Cav3.2 T-type calcium channel can physically and functionally interact with the BKrization . BKCa exc-fibers .ASCI was higher in patients with CKD and H+ sensitivity than in healthy individuals, but the difference was not statistically significant, suggesting no direct impact of pH on CKD-aP.Many times in the past a new treatment option has been reported to be effective, but very soon thereafter conflicting results appear. Most therapeutic trials have shown only limited success. We think that this is because pruritogens mainly act on ion channels indirectly through many GPCRs or ROS and do not act on ion channels directly such as on pain stimuli.This cohort-sectional study had some limitations. First, the total number of samples were relatively small, which may restrict interpretation of our results. Due to the small number of subjects, it was divided into two groups (non-CKD-aP and CKD-aP) instead of five groups . However, this separating way may be less meaningful, because each pruritus has different molecular profile.Second, we examined ion channels specifically expressed in peripheral nerve endings  and those expressed in keratinocytes or other sites, as well as peripheral nerve ending . Presently, it is technically difficult to separate the peripheral nerves from skin cells and examine ion channels expressed only the peripheral nerve endings. This requires further research. We think that the combination of ion channel agonists and antagonists will lead to new drug discoveries in the future. In addition to expression studies, functional studies are also needed to investigate the physiological roles of these channels. There are many reports that increased membrane expression of the ion channel parallels functional up-regulation of the ion channel in neurons .It was concluded that a greater difference was observed in expression of ion channels at the skin tissue including, specific, for cutaneous peripheral nerve endings in CKD patients with CKD-aP than in those without CKD-aP. This up-regulated expression of Cav3.2 T-type calcium channel of the peripheral nerve in patients with CKD-aP may increase the generator potential and induce action potentials related to itching.


Since the clear demonstration of lysophosphatidic acid (LPA)’s pathological roles in cancer in the mid-1990s, more than 1000 papers relating LPA to various types of cancer were published. Through these studies, LPA was established as a target for cancer. Although LPA-related inhibitors entered clinical trials for fibrosis, the concept of targeting LPA is yet to be moved to clinical cancer treatment. The major challenges that we are facing in moving LPA application from bench to bedside include the intrinsic and complicated metabolic, functional, and signaling properties of LPA, as well as technical issues, which are discussed in this review. Potential strategies and perspectives to improve the translational progress are suggested. Despite these challenges, we are optimistic that LPA blockage, particularly in combination with other agents, is on the horizon to be incorporated into clinical applications. The curt cancer ,47,48,49The major challenges in moving LPA targeting to clinical practice include the complex metabolic network of LPA, the extremely broad and multifaceted pathological activities elicited by LPA, which are overlapping with its physiological activities, the complicated and potentially opposing cellular activities mediated by different LPA receptors in an individual cancer and individual patient-dependent manner, and the maze of intertwined G-protein coupling and downstream signaling pathway elicited by LPA through its own receptors, as well as many other types of cell receptors and signaling pathways. Moreover, technical issues for LPA detection and/or blockage, as well as study design issues are also major obstacles to overcome. This review focuses on these issues with perspectives to improve the LPA translational progress.LPA was first described in the early 1960s ,51. It wLPA was first isolated from brain extracts in 1961 . Most stDictyostelium discoideum amoebae and human neutrophils [The 1980s were the beginning decade for extensive functional and signaling studies of LPA. LPA-induced platelet aggregation and alterations in arachidonate metabolism were the earliest LPA functions revealed, which were further studied over the following decades ,103,104.trophils ,116. Mortrophils ,118,119.2+ mobilization and stimulation of protein kinase C; release of arachidonic acid in a guanosine triphosphate (GTP)-dependent manner, but independent of prior phosphoinositide hydrolysis; and activation of a pertussis toxin-sensitive Gi-protein mediating inhibition of adenylate cyclase [In terms of signaling properties, calcium and cyclic adenosine monophosphate (cAMP) are the earliest revealed downstream signaling molecules for LPA . Its reg cyclase . Later,  cyclase ,119. Ano cyclase .The implications of the potential roles of LPA in cancer stem from the findings for LPA’s mitogen- and growth factor-like activity in the late 1980s and early 1990s ,57,58,59Although G-protein-mediated LPA signaling pathways were identified as described above, molecular identification and cloning of LPA receptors in 1996 and the following years established the corner stones for rapid growth of LPA-related studies and targeting, as G-protein coupled receptors (GPCRs) represent targets for ~40% of pharmacological drug antagonists .1/EDG2, LPAR2/EDG4, LPAR3/EDG7, LPAR4/purinergic G protein-coupled receptor P2Y9  cells, mainly via PPARγ, and LPA’s cancer stem cell (CSC)-promoting activities are mediated by PPARγ [The first LPA receptor was identified and cloned in 1996 , which wAR6/P2Y5 ,69,70,71AR6/P2Y5 ,125, GPRAR6/P2Y5 , and P2YAR6/P2Y5 . HoweverAR6/P2Y5 ,130,131.AR6/P2Y5 , and theAR6/P2Y5 . LPA wasAR6/P2Y5 . Until rby PPARγ .Another important milestone in LPA research was the identification of the major LPA-producing enzyme, autotaxin (ATX). Although the enzymatic activity of the lysophospholipase D in the production of LPA was described earlier in rat plasma , the genThe crystallization and structure determination for LPA GPCRs belonging to each of the two subclasses (EDG and purinergic receptors), as well as ATX ,92,93,94The functions/cellular effects of LPA  are very broad, which were extensively reviewed ,137,138.We initially reported LPA as a potential marker for ovarian cancer (OC) , which iHowever, we are facing several challenges in moving LPA as a cancer marker to clinical application . These iThe analytic methods for LPA were greatly advanced from earlier (1960–1990s) thin-layer and high-performance liquid chromatography-based analyses ,142,143 To overcome these obstacles, one possibility is to measure the levels of ATX, the key enzymes producing LPA ,89, suchIt may be critical to develop more direct detection methods for LPA from human samples, such as a drop of fingertip blood on a filter paper, to avoid effects derived from variations in lipid extraction and storage conditions. Direct imaging/reporter-based methods may represent another direction to bypass the sample handling related artefacts.As mentioned above, LPA represents a group of compounds varying in their chemical linkage to the glycerol backbone, number of carbons, and number and location of double bonds, with their molecular weights between 400 and 500 Da ,149. In 2 enzymes (sPLA2s) [2s [2s are not only critical in generating the substrates for ATX to produce LPA, but they also generate LPA directly by acting on phosphatidic acid as its substrate [2s, at least 10 were studied in cancer, with most of them being aberrantly expressed in cancer [LPA production and catabolism are controlled by a complex network of enzymes. Extracellular LPA is mainly produced by ATX and soluble phospholipase A(sPLA2s) . Other PA2s) [2s ,165,166 A2s) [2s ,168 playubstrate ,164,165.n cancer  , which was recently reviewed extensively ,95,135. 2+, protons, and photons [GPCRs are the largest superfamily of receptors, with the identification of 865 human GPCR genes . Compare photons . GPCRs a photons . The maj6 and double G-protein coupled LPAR3 to couple to both G12/13 and Gi for LPAR6 [13, in addition to Gq and Gi for LPAR3 [One of the challenges in targeting LPA GPCR receptors is their complex array of G-protein coupling, resulting in multi-faceted outcomes. While most of the other individual GPCRs, including most of the best-studied β-adrenergic receptors, neurotransmitter receptors, and sensor GPCRs  ,183,184 or LPAR6 ,186,187 or LPAR3 ,189  or a potentially Gs-independent pathway to increase cAMP via LPAR5 [5 were also shown to enhance cell proliferation and motility in rat lung and liver cancer cells [q and/or G12/13 [5 in cytotoxic T cells may actually have a pro-tumorigenic effect [While tumor-promoting activities are more consistently associated with LPAR pathway , LPAR4–6ectively . LPAR4 rus cells . In panctivities . LPAR5 ial cells . These iia LPAR5 . Contrader cells , which mr G12/13 . On the c effect  , receptors with other enzymatic activities (serine or serine/threonine kinases and guanylyl cyclase enzymatic activities), other GPCRs, integrins, cytokine receptors, and T- and B-cell receptors, as well as intracellular receptors, such as PPARγ. Listed below are examples from these categories .2+ and Ca2+-activated potassium channels, and the Na+/H+ exchanger 3 (NHE3) via the LPAR5 receptor, which also involves the epidermal growth factor receptor (EGFR) [γ [LPA stimulates and regulates several ion channels, including the Car (EGFR) ,211. LPAr (EGFR) . We receEGFR) [γ  –GPCR signal complexes is a focal point for the study of integration of cell signaling, which plays an important role in signal transduction . The croLPA inhibits the natriuretic peptide-induced generation of cGMP via a non-receptor tyrosine kinase Csk ,229,230.13/p115RhoGEF/RhoA pathway and the β2-adrenergic receptor/Gs/adenylyl cyclase pathway was reported [LPA interacts with other GPCR receptors. Free fatty-acid receptors  have a potential negative cross-talk between LPA receptors and EGF receptors ,236. LPAreported . LPA alsreported . At physreported .1 is an inducer of S1PR1/β-arrestin coupling. This interaction promotes the porous junctional architecture of sinus-lining lymphatic endothelial cells and enables efficient lymphocyte trafficking [2 and the small Gq [1 in mammalian cells [There are close interactions between two oncogenic lysolipids, LPA and S1P, in their overlapping signaling pathways and/or directly in their receptors . These tfficking . The funfficking . Lamininfficking . LPA indsmall Gq . LPA uprsmall Gq ,251, andan cells . Moreovean cells  and intean cells . Most noan cells ,255, proan cells   signaling [5 also inhibits B-cell receptor (BCR) signal transduction via a Gα12/13/Arhgef1 pathway [LPARignaling . LPAR5 a pathway . On the  pathway .The cross-talk can be mono- or bidirectional and can be either positive or negative cross-talk, dependent on the type of interaction, the cell types, and the biological effects involved . For exaIt is important to note that LPA is involved in several stem cell/cancer stem cell (CSC) signaling pathways. The ATX–LPA signaling pathway is recognized as a critical new player in CSC . LPA is 1–3 and GPR4. Interestingly, it was shown that LPA remarkably enhances, through the LPAR1/Gi protein, the OGR1-mediated vascular actions to acidic pH [LPA cross-talks with other signaling molecules at many different levels with divergent mechanisms. Firstly, interactions are through direct binding/interactions. Homo- and heterodimerization of LPA/S1P receptors, ovarian cancer G protein coupled receptor-1 (OGR1) and GPR4, was shown using LacZ complementation assays . LPA reccidic pH . These rcidic pH   and a disintegrin and metalloproteinase (ADAM)-catalyzed membrane shedding of heparin-binding EGF and autocrine/paracrine activation of EGF ,268,269,OCT4, SOX2, SOX9, ALDH1, and drug transporters [Thirdly, an LPA-regulated transcriptome is involved. LPA regulates many cytokines, including IL-6, IL-8, growth-regulated oncogene (GRO)-α ,270,271,sporters ,275,276,Fourthly, downstream signaling pathway interactions play important roles. The signaling pathways involved in LPA cross-talk include, but are not limited to the PI3K/Ras , the mitFinally, other signaling molecules may regulate metabolic enzymes for LPA and other lipid molecules. Neurotransmitters, cytokines, and growth factors regulate the activity of a key set of lipid-metabolizing enzymes, such as phospholipases, to affect LPA and other lipid signaling molecules . In addiThe targeting of one or more of these cross-talks and/or the major LPA downstream signaling pathways may be critical and/or more efficient than targeting LPA or LPAR directly. For example, the FDA recently approved the first PI3K inhibitor for breast cancer treatment. The challenges are identifying one or more driver targets at the level of individual cancer type and individual patient.2 (PLA2), which is a soluble PLA2, produces extracellular LPA and is involved in EOC and associated with early relapse of EOC [Targeting the tumor-prone microenvironment gained increasing attention in recent years . Althouge of EOC ,285 , 3LPAR (a LPA receptor), SMPD1 (sphingomyelin phosphodiesterase 1 that converts sphingomyelin to ceramide), PPAP2B  emerged as the most prognostically important markers. In particular, alignment of data across a variety of malignancies (over 600 different neoplasm categories) revealed specific preference for ovarian carcinoma [1, and LPAR5 are higher in the immune-high tumor  group, but LPAR2.3 are higher in the immune-low group [While S1P’s functions in the immune system were extensively studied, and the S1P receptor axis represents an obligatory signal for trafficking of immune cells , the roliltrates . These wglecerol ), and SMarcinoma . It is iow group , suggesthttps://www.cell.com/pb-assets/consortium/ pancanceratlas/pancani3/index.html?code=cell-site). One such study related to LPA identified novel sphingolipid–LPA immune checkpoints and networks underlying tumor immune heterogeneity and disease outcomes, as mentioned in the While LPA and/or the ATX–LPA axis are generally accepted as important targets for cancer, we are still facing several major obstacles to move targeting to clinical practice as presented above. Personalized medicine is now well accepted conceptually to treat highly heterogenic diseases, such as cancers. The complex LPA metabolism, its receptor, and its signaling systems suggest that detecting signatures/networks, rather than individual gene/protein/lipid expression, from individual patients will likely become necessary to develop effective treatments for highly heterogenic diseases, such as cancer. Bioinformatics/systems biological analyses become very important to achieve such goals, which provide unprecedented scale and depth of knowledge and perspectives in cancer research, as well as directions for translational and clinical applications. These studies use multi-modular integrative approaches consolidating large amount of data from gene expression profiling, next-generation sequencing, -omics studies, prognostic/predictive modeling, and functional studies for cancer ,296. TheIt is important to note that, although targeting LPA signaling appears to be an attractive approach for cancer treatment, it is unlikely that this will be effective as monotherapy. One of the major challenges in LPA targeting will be to discover the magic modalities to be effective in cancer treatment, which are likely to be different for different cancers and even for different individuals. Synthetic lethality studies led to the clinical benefits of using poly (ADP-ribose) polymerase (PARP) inhibitors in OC and other cancers, which is a very significant step forward in the field . SimilarTaken together, although we are still facing a number of obstacles, the large body of data generated from the last 3–4 decades from thousands of papers provided solid evidence and rationale to target the LPA network. Rapid development of biotechnological tools for analyses of LPA, as well as its functions and mechanisms, and for processing the vast amount of data provided and will continue to furnish essentials for the field to leap ahead and eventually improve patient outcomes.