S176 is a critical post-translational modification that suppresses YAP’s ability to promote cell proliferation and tumorigenesis by altering the subcellular localization of an essential YAP co-factor.The Hippo-YAP pathway is a central regulator of cell contact inhibition, proliferation and death. There are conflicting reports regarding the role of Angiomotin (Amot) in regulating this pathway. While some studies suggest a YAP-inhibitory function other studies indicate Amot is required for YAP activity. Here, we describe an Amot-dependent complex comprised of Amot, YAP and Merlin. The phosphorylation of Amot at Serine 176 shifts localization of this complex to the plasma membrane, where it associates with the tight-junction proteins Pals1/PATJ and E-cadherin. Conversely, hypophosphorylated Amot shifts localization of the complex to the nucleus, where it facilitates the association of YAP and TEAD, induces transcriptional activation of YAP target genes and promotes YAP-dependent cell proliferation. We propose that phosphorylation of AmotDOI:http://dx.doi.org/10.7554/eLife.23966.001 Cells in animals and other multi-cellular organisms need to know when and where they should grow and divide. Individual cells communicate with their surrounding environment and each other via signaling pathways such as the Hippo-YAP pathway, which stimulates cells to grow and therefore influences the size of organs. When the Hippo part of the pathway is active it causes a protein known as YAP to move out of a compartment in the cell called the nucleus. Inside the nucleus, YAP helps to activate genes that promote cell growth. If the Hippo pathway can no longer respond to cues from the environment, YAP becomes over-active and can contribute to the development of various cancers. Therefore researchers are trying to better understand how it is regulated.Many signals both from inside and outside the cell influence YAP activity. For example, some signals block YAP from entering the nucleus, whereas others cause YAP to be broken down entirely. Several studies have recently identified a signal protein called angiomotin as a regulator of YAP. However, the studies provide conflicting reports as to whether angiomotin promotes or inhibits cell growth.Like many other proteins, angiomotin can be tagged with a small molecule called a phosphate group that can alter its activity. Moleirinho, Hoxha et al. studied human cells containing versions of angiomotin that mimic different forms of the protein with or without the phosphate. The experiments indicate that when a phosphate is attached at a particular position (known as serine 176), angiomotin predominantly interacts with YAP and another protein called Merlin at the cell surface. On the other hand, when angiomotin does not have a phosphate attached to it, all three proteins can move into the nucleus, where YAP is able to activate genes and promote cell growth.Overall, these findings indicate that adding a phosphate group to angiomotin can act as a switch to regulate where in the cell it and YAP are found and thus, whether YAP is active. Future experiments will investigate which enzymes add the phosphate group to serine 176, and when they are able to do so.DOI:http://dx.doi.org/10.7554/eLife.23966.002 Angiomotin (Amot) was originally identified as an angiostatin-binding protein involved in the regulation of endothelial cell polarization, migration, proliferation, and angiogenesis . Amot is106LPTY109/239PPxY242 motifs present in the N-terminal domain of Amot-p130 and WW domain present in YAP  mice showed Amot is required for hepatic ductal cell proliferation and tumor formation in the context of either Nf2 loss or DDC -induced injury. Additionally, substantially increased expression of Amot was observed in NF2-null human schwannomas samples, which primarily displayed localization of Amot in the nucleus  as a binding partner of YAP and Merlin . We firsAs previous studies reported multiple cellular localizations for Merlin and YAP, we sought to determine the cellular compartments where YAP and Merlin co-localize. Flag-tagged Merlin and V5-tagged YAP were co-transfected in both HEK293 and hSC2λ cells and the localization and association of these proteins were examined by subcellular fractionation coupled to IP with an anti-Flag antibody. These subcellular fractionation studies showed an association between Merlin and YAP primarily in the nucleus and cytoplasm, and to a much lesser extent at the plasma membrane . FurtherSince Amot directly binds YAP and Merlin through two distinct domains , 2011, w239PPEY242 and 284PPEY287), and an unconventional LPTY motif (106LPTY109) that mediate the interaction with YAP  or HA-Amot-p130S176E (phosphomimetic mutant) and similar levels of the different Amot alleles were confirmed  . We theronfirmed . IPs weronfirmed . Furtheronfirmed . In addionfirmed . CollectS127A mutant retain their ability to associate with Amot/Merlin  in these cells compared to control 293-shAmot cells. As expected, transfection with the different Amot alleles resulted in increased Rac1-GTP levels. However, we found no significant differences in Rac1-GTP levels between the cells, suggesting the status of Amot serine 176 phosphorylation does not affect Rac1 activation  and ApoE (Apolipoprotein E) as previous findings suggest Amot plays a role in their regulation  is recruited to the plasma membrane where it is found co-localized and associated with Patj, Pals1 and E-cadherin at junctional structures. Our previous studies indicate that Amot is required for YAP function in the nucleus (S176A) is preferentially localized to the nucleus, where it facilitates YAP interactions with TEADs and concomitant activation of target gene transcription.The role of Angiomotin regulating the Hippo/YAP pathway has so far been elusive, mainly due to conflicting reports suggesting that YAP regulation by Angiomotin is either positive or negative. To gain a deeper understanding of Amot function, we employed multiple cell types originating from tissues in which dysregulation of the Hippo-YAP pathway is observed under pathological conditions. Our study reveals that Angiomotin phosphorylation at serine 176 mediates YAP localization. This post-translational modification is the key event determining a promoting or repressing regulation of YAP by Angiomotin. We found that phosphorylation at serine 176 does not mediate Angiomotin’s scaffolding functions but does impact the localization of a tertiary complex composed by Amot, YAP and Merlin, which is present in both the cytoplasm and nucleus for the wild type protein. The serine 176 phosphomimetic mutant . The following plasmids were previously described: psCMV-Pals1-Flag; PATJ-Flag . All experiments were carried out on cells grown to 70–80% confluency. Transfections were performed using Lipofectamine 2000  unless stated otherwise. Lentiviral infection of HEK293 cells was performed according to standard protocols. Briefly, HEK293T cells were co-transfected with packaging plasmids VSVG, Δ8.2, and either with pLKO.1-GFP or pLKO.1-shAmot constructs. Supernatants were collected 48 hr and 72 hr after transfection, and cells were infected with 4 mL of viral supernatant containing 4 mL of polybrene (8 μg/mL). After 48 hr, transduced cells were selected with puromycin (2 μg/mL) and this selection maintained for 72 hr.HEK293 and HepG2 cells were purchased from the ATCC. hSC2λ cells were obtained from the laboratory of Dr. Margret Wallace . All cel+/K+ATPase  from Developmental Studies Hybridoma Bank (University of Iowa). For immunofluorescence the following antibodies were used at the stated concentrations: rabbit monoclonal anti-YAP D8H1X , Cell Signaling Technologies; mouse monoclonal anti-Merlin , Santa Cruz Biotechnologies; Rabbit polyclonal anti-Angiomotin (1: 200).Rabbit polyclonal anti-Angiomotin was previously described  . Eluted proteins were separated on SDS/10% polyacrylamide gel and transferred onto Immobilon-P membranes (Millipore). To prevent nonspecific binding, membranes were incubated in blocking buffer  with agitation for 1 hr at room temperature, followed by immediate incubation with specific antibodies diluted in either 5% BSA or blocking buffer, overnight. Membranes were then washed three times in washing buffer , incubated for 1 hr at room temperature with goat anti-mouse HRP-conjugated antibody  or Protein A-HRP linked  and protein expression was detected by chemiluminescence using ECL .150 cm diameter dishes were transiently transfected with 10 μg of plasmid DNAs using Lipofectamine 2000 (Invitrogen). 48 hr later cell lysates were collected, washed twice in ice-cold PBS, and lysed with radioimmuno precipitation assay buffer ). 1 mg of protein was immunoprecipitated with Protein A/G resins  and indicated antibody with gentle rotation at 4Cells were collected and lysed with actin stabilization buffer . Biotinylated-phalloidin  was added to samples and incubated for 60’ at 4°C with constant rotation. Subsequently, 20 µL of streptavidin-coupled magnetic Dynabeads were added, incubated for 60’ at 4°C with constant rotation. The magnetic beads were isolated with a magnet, washed 3X with PBS and analyzed as described.Immunofluorescence staining was carried out as previously described . Briefly2, 1 mM PMSF, 1 mM DTT, 0.5%Nonidet P-40, protease inhibitor mixture), centrifuged at 4000 g for 5’ at 4°C. Supernatant was kept for the cytosolic and plasma membrane fractions. The pellet was washed five times with nuclear washing buffer , lysed on ice for 20 min in 400 μl of RIPA buffer and centrifuged at 13,000 g for 10 min at 4°C. Supernatant was kept as the nuclear fraction. For the cytosolic and plasma membrane fractions the supernatant was spun at 200,000 g for 30 min at 4°C and the resultant supernatant centrifuged again at 13,000g for 5 min at 4°C and kept as the cytosolic fraction. The pellet was washed in 500 μl lysis buffer  and re-sedimented at 200,000g for 30 min at 4°C. The pellet was then resuspended in ice-cold IP buffer  Triton X-100, and complete protease inhibitor mixture), incubated on a rocker for 30 min at 4°C, and cleared by centrifugation at 13,000g for 15 min at 4°C. The supernatant was kept as the plasma membrane fraction.Cells were incubated on ice for 20 min in ice-cold cytoplasmic buffer  and 48 hr later lysed in RIPA buffer and precipitated with indicated antibody overnight. Rac1-GTP was pulled down according to the manufacturer’s instructions .HEK293-shAmot cells were transfected with 8 μg of plasmid DNA (empty vector control/ wild type Amot-p130/Amot5 cells per well in 6-well plates. On the following day cells were co-transfected with 40 nM of siRNAs (targeting either YAP or a non-targeting siRNA duplex (control)), and 2 μg of plasmid DNA (empty vector control/ wild type Amot-p130/AmotS176A/AmotS176E) using TransIT-LT1 and TransIT-TKO  according to manufacturer’s instructions. After 24 hr, cells were trypsinized and 2 × 104 cells/well were reseeded in sterile 96-well tissue culture plates. 48 hr, 72 hr, and 96 hr after co-transfection, plates were processed and analysed using BrdU cell proliferation assay kit following manufacturer’s instructions . Plates were read using a spectrophotometer microplate reader set to 450 nm .HEK293-shAmot cells were seeded at a cell density of 2 × 103 cells/50 μl in 96-well plates. On the following day the indicated luciferase reporters and plasmids were transfected to a total of 160 ng and incubated overnight at 37°C. Luciferase activity was then measured with Dual Luciferase Reporter Assay System  according to manufacturer’s instructions. The reporter’s firefly luciferase activity was normalized to the levels of Renilla luciferase used as an internal control reporter. The relative luciferase activity displayed on the Y-axis indicates the ratio between Firefly/Renilla luciferase activities.The HIP/HOP-flash luciferase reporter system was a kind gift from Dr. Barry Gumbiner  and the −ΔΔCT algorithm (Extraction of RNA from cell lysates was performed using Qiagen RNeasy kit (Qiagen) followed by cDNA synthesis of 1 μg DNase-digested RNA, using SuperScript III First-Strand Synthesis System for quantitative RT–PCR (Invitrogen) according to manufacturer’s instructions. Quantitative PCR of the synthesized cDNA was conducted using SYBR Green 2x Master Mix (Applied Biosystems) according to the manufacturer’s protocol. 10 ng of each sample were used in each analysis. Real-time quantitative RT-PCR reactions were performed on StepOnePlus Real-Time PCR System (Applied Biosystems) and analysed using StepOne Software v2.2.2. All measurements were conducted three times in triplicate and standardized to the levels of GADPH. Relative changes in gene expression were calculated according to the 2lgorithm . Sequenc20 million HEK293T-shAmot cells were fixed with 1% formaldehyde for 10 min at RT. Fixation was halted with 125 mM glycine for 5 min at RT. Fixed cells were washed twice with cold PBS. Cell pellets were then resuspended in ChIP lysis buffer and chromatin was sheared with sonicator to obtain 0.3–0.5 kb DNA fragments. Angiomotin antibody (5 μg) and Dynabeads Protein A were added to the cell lysate and incubated overnight at 4°C. Beads were washed with buffer 1  and then washed with TE buffer. DNA was eluted by resuspending the beads in TE/1%SDS. ChIP DNA and Input were treated with RNase A (5 μg) for 1 hr at 37°C. Proteinase K (0.5 mg/mL) was added and incubated overnight at 65°C to reverse crosslinking. DNA was then purified phenol:chloroform and resuspended in a 30 μL of elution buffer. DNA was used for real time-PCR using SYBR Green PCR kit. A standard dilution curve was obtained for each Input and 1 μL of ChIP DNA was used in each PCR reaction. Melt curves were analyzed to confirm specificity of the amplified target. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by Anna Akhmanova (Senior Editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors. The reviewers have opted to remain anonymous.Thank you for submitting your article "Regulation of localization and function of the transcriptional co-activator YAP by angiomotin" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.In this manuscript, the authors demonstrate that Amot acts as a scaffold to recruit YAP to Merlin, and that the formation of this complex is not affected by the phosphorylation of Amot at S176. Interestingly, however, the mutation of S176 to E induces a clear relocalization of Amot from the nucleus to the plasma membrane and the cytosolic fraction , while mutation to A result in strong nuclear signal. This is accompanied by a redistribution of YAP and Merlin (and in consequence of the complex) to the plasma membrane in the S176E cells, perhaps through augmented association with Pals1/Patj. The consequence of the phosphorylation status of Amot on proliferation / YAP activity was investigated, revealing strong activity of the S to A mutant in all assays, and inactivity of the S176E mutant. It provides evidence that AMOT facilitates YAP nuclear and transcriptional function, unless phosphorylated at ser 176, which facilitates AMOT interaction with plasma membrane proteins. The authors’ findings disagree with some previous conclusions about the role of AMOT, which they address well, and which makes the finding of importance.All reviewers thought that this work was novel and interesting. However additional controls are considered necessary to demonstrate some of the reagents are specific, quantification is needed, and some of the conclusions should be moderated or caveats considered in the Discussion.1) What is the expression level of the ectopically expressed Amot, compared to that endogenously expressed in HEK cells? Almost the entire analysis depends on exogenous expression of AMOT and AMOT mutants. It is well known that overexpression of proteins often leads to their accumulation in inappropriate compartments. Since so much of the conclusions and model depend on the idea of compartments and pools, it is essential that the authors ensure that their proteins are expressed at normal cellular levels, and that all mutants are expressed at similar levels2) Ectopically expressed PATJ and PALS1 co-Ips with Amot and AmotS>E, but not AmotS>A. This may be due to Amot localization at the membrane versus nucleus. Is association of Amot with Patj/PALS1 really needed for the membrane localization of the complex? The authors should knockdown Patj/PALS1  to support this conclusion.3) Could the interaction (or non-interaction) of the Amot constructs with Patj and Pals1 be only a reflection of the differential proportion of the Amot constructs in the plasma membrane fraction where Patj and Pals1 reside? . As presented, this set of experiments is supportive of the differential localization, but does not necessarily demonstrate a direct role for association with Patj/Pals1 . Is the association with Patj/Pals1 bringing Amot to the cortex? If more direct evidence for a preferential association of Amot S176E to Patj/Pals1 cannot be experimentally determined, the authors may want to revise the presentation of the associated results and conclusions.Specific comments/suggestions to address in the Discussion:4) Is it possible that overexpressed protein does not reveal a change in affinity upon altered phosphorylation?5) The authors should discuss the 5A YAP results. Do they believe this can be explained by increased nuclear localization of 5AYAP?6) The authors ignore the role of Lats, which is also thought to bind to AMOT, and the role of its phosphorylation of YAP in YAP regulation. Most studies on the hippo pathway agree that this phosphorylation event controls both the levels and nuclear-cytoplasmic distribution of YAP, but these two properties are not addressed. The authors do show that phosphorylation of YAP on ser 127 does not affect binding to AMOT, but do not show how AMOT regulates YAP phosphorylation or stability. Also, they do in fact see a change in binding of 5 ser > ala YAP mutations, which have been shown to regulate YAP function in addition to s127. This difference must be considered.7) There is no consideration of any upstream regulation of the pathway in either the experimental analysis or in the evaluation of the models and literature. What controls AMOT phosphorylation? Are Mst/hippo kinases involved in AMOT regulation? Are the authors imagining that this regulation has something to do with cell-cell contact control of the pathway? Or control by soluble growth factors and signaling receptors? In the absence of this broader context, it is very difficult to think about mechanisms and models. At the least this should be touched on in the Discussion.8) Their model implies a constitutive binding of YAP to AMOT in the control YAP localization by AMOT, without considering catalytic effects, especially those that might be conferred by Lats phosphorylation. Similar to other models, it invokes simple sequestration at the membrane via AMOT-PATJ binding to restrain YAP. Yet only a small fraction of S176 AMOT seems to associate with membranes or PATJ. Similarly, the model implies that the cytoplasmic and nuclear pools of YAP are all bound to AMOT. If a large fraction of YAP in the cytosol or nucleus is free, different mechanisms from the one proposed must be involved in controlling its localization.9) The authors discuss previous studies showing that AMOT interacts with the actin cytoskeleton and that this interaction is regulated by AMOT phosphorylation. However, they do not address this potentially important pool of AMOT in their study. How does the actin associated pool fractionate in their protocol – with cytoplasmic, nuclear, or membrane fractions?10) 11) Others have reported that cadherins can also interact with merlin-AMOT complexes at the membrane in addition to PATJ. Do the authors think that this may also be a significant interaction worth examining?12) The interpretation that the interaction between YAP and Merlin is predominantly occurring in the nucleus and the cytoplasm is supported by the data, but quantification of these experiments would help providing an assessment as to whether this simply reflect the relative levels of YAP across the different fractions or whether there is evidence for a truly differential interaction across fractions. . Perhaps an alternative way to provide support for a compartment-specific interaction would be to employ a fluorescence-based approach .13) The presentation of the western blots (IP/western) should be accompanied by quantification. I was surprised that experiments that I would assume should be loaded on the same SDS page gel (e.g. different constructs for the rescue experiments in 14) What is the intracellular localization of endogenous Merlin (and YAP)? The localization experiments currently presented all depend upon transient transfection.15) For 16) If the phosphospecific antibody works in IF, it would be nice to provide additional evidence that the phosphorylated form is enriched in the plasma membrane fraction . This would support the results obtained with the S to E mutation.17) What is the relative level of expression (in relation to the endogenous) of the different Amot constructs? Similarly, the authors may want to comment on the expression levels of the other constructs they used in relation to the respective endogenous proteins. Could some of the results observed due to overexpression / tagging artifacts?18) Concerning the 19) Other comments: – this likely happened through PDF generation during submission, but the quality of the images is problematic. Labels do not display clearly, and microscopy images are extremely difficult to evaluate. The authors need to provide high quality images with the revision. […] All reviewers thought that this work was novel and interesting. However additional controls are considered necessary to demonstrate some of the reagents are specific, quantification is needed, and some of the conclusions should be moderated or caveats considered in the Discussion.1) What is the expression level of the ectopically expressed Amot, compared to that endogenously expressed in HEK cells? Almost the entire analysis depends on exogenous expression of AMOT and AMOT mutants. It is well known that overexpression of proteins often leads to their accumulation in inappropriate compartments. Since so much of the conclusions and model depend on the idea of compartments and pools, it is essential that the authors ensure that their proteins are expressed at normal cellular levels, and that all mutants are expressed at similar levelsWe agree with the reviewers’ comments. Assessment of expression of the different Amot alleles and comparison to endogenous Amot levels are now included in 2) Ectopically expressed PATJ and PALS1 co-Ips with Amot and AmotS>E, but not AmotS>A. This may be due to Amot localization at the membrane versus nucleus. Is association of Amot with Patj/PALS1 really needed for the membrane localization of the complex? The authors should knockdown Patj/PALS1  to support this conclusion.In spite our extensive attempts, using several different shRNA/siRNAs, we were unable to achieve efficient knockdown of PATJ/PALS1 and thus unable to assess whether the membrane localization of AmotS>E is changed upon loss of these proteins. In addition, while responding to other review comments, we are able to detect enhanced association of the AmotS>E mutant with E-cadherin . Therefo3) Could the interaction (or non-interaction) of the Amot constructs with Patj and Pals1 be only a reflection of the differential proportion of the Amot constructs in the plasma membrane fraction where Patj and Pals1 reside? . As presented, this set of experiments is supportive of the differential localization, but does not necessarily demonstrate a direct role for association with Patj/Pals1 . Is the association with Patj/Pals1 bringing Amot to the cortex? If more direct evidence for a preferential association of Amot S176E to Patj/Pals1 cannot be experimentally determined, the authors may want to revise the presentation of the associated results and conclusions.Please see our response to question 2.Specific comments/suggestions to address in the Discussion:4) Is it possible that overexpressed protein does not reveal a change in affinity upon altered phosphorylation?While this is a possibility, we believe the likelihood for this is low. If one hypothesizes that phosphorylation at S176 has an impact on the affinity of Amot towards merlin or YAP, it would be expected that the S176A or S176E mutants would have different effects on affinity and association with their binding partners, which is not what we observed in our studies.5) The authors should discuss the 5A YAP results. Do they believe this can be explained by increased nuclear localization of 5AYAP?The literature indicates the S5A mutant is preferentially localized to the nucleus. The experiment shown in 6) The authors ignore the role of Lats, which is also thought to bind to AMOT, and the role of its phosphorylation of YAP in YAP regulation. Most studies on the hippo pathway agree that this phosphorylation event controls both the levels and nuclear-cytoplasmic distribution of YAP, but these two properties are not addressed. The authors do show that phosphorylation of YAP on ser 127 does not affect binding to AMOT, but do not show how AMOT regulates YAP phosphorylation or stability. Also, they do in fact see a change in binding of 5 ser > ala YAP mutations, which have been shown to regulate YAP function in addition to s127. This difference must be considered.Although we did briefly discuss the role of Lats in the paper, the role of Lats is complicated and not straightforward. Indeed Lats1/2 phosphorylate and regulate YAP localization and levels and a number of studies have shown Lats regulates phosphorylation of Amot on S176. However, the relationship between Lats, YAP and Angiomotin is complex. For example, a number of papers suggest that Lats phosphorylates Amot, leading to reduced binding to F-actin and increased YAP binding and inhibition . Moreover, Amot has been proposed to activate Lats2 and increase phosphorylation of YAP . In contrast, we have previously shown that in multiple systems, Amot antagonizes the association of Lats and YAP and inhibits phosphorylation of YAP by Lats . Given the complexity of the interactions between YAP, Amot and YAP, we believe this question requires a more extensive and in depth analysis which is beyond the scope of the current manuscript. Some of the issues raised in our response are also addressed in the Discussion of the manuscript.7) There is no consideration of any upstream regulation of the pathway in either the experimental analysis or in the evaluation of the models and literature. What controls AMOT phosphorylation? Are Mst/hippo kinases involved in AMOT regulation? Are the authors imagining that this regulation has something to do with cell-cell contact control of the pathway? Or control by soluble growth factors and signaling receptors? In the absence of this broader context, it is very difficult to think about mechanisms and models. At the least this should be touched on in the Discussion.We agree with this point. We initially left this out due to space considerations. In particular, while are many possible scenarios involving regulation of Amot activity by upstream events, since S176 has been shown to be phosphorylated by Lats, we can speculate that many of the upstream events regulating the Hippo cascade will also regulate Amot. We now include this in the Discussion.8) Their model implies a constitutive binding of YAP to AMOT in the control YAP localization by AMOT, without considering catalytic effects, especially those that might be conferred by Lats phosphorylation. Similar to other models, it invokes simple sequestration at the membrane via AMOT-PATJ binding to restrain YAP. Yet only a small fraction of S176 AMOT seems to associate with membranes or PATJ. Similarly, the model implies that the cytoplasmic and nuclear pools of YAP are all bound to AMOT. If a large fraction of YAP in the cytosol or nucleus is free, different mechanisms from the one proposed must be involved in controlling its localization.We agree that examining the dynamics of the association of the complex would be useful, however, given space and time limitations we did not yet get to do these types of analyses.We agree that a fraction of Amot-S176E appears to be localized to the PM, while additional protein is found in the cytoplasm and none in the nucleus (see example in 9) The authors discuss previous studies showing that AMOT interacts with the actin cytoskeleton and that this interaction is regulated by AMOT phosphorylation. However, they do not address this potentially important pool of AMOT in their study. How does the actin associated pool fractionate in their protocol – with cytoplasmic, nuclear, or membrane fractions?We thank the reviewer for this comment. In the revised version of the manuscript we include data assessing the association of the different Amot alleles with F-actin. While most of the other reports relied on co-localization by IF and in vitro studies using purified protein, we wished to directly assess the interaction of Amot with F-actin in cells. To achieve this, we used biotin-XX-phalloidin which preferentially binds F-actin. We then precipitated the F-actin from cells transfected with the different Amot mutants and assessed the levels of Amot that were co-precipitated. This analysis shows that there are no differences in the association of the different mutants with F-actin. While our findings contrast with some of the previous reports regarding the association of Amot with F-actin, we believe that the approach we employed directly assess the interaction between F-actin and Amot, while other reported efforts employed methods that are either indirect (IF) or in vitro. We include this new data in 10) We thank the reviewer for catching this. We now provide a revised figure in which the levels of IPed PATJ are similar between the different conditions.11) Others have reported that cadherins can also interact with merlin-AMOT complexes at the membrane in addition to PATJ. Do the authors think that this may also be a significant interaction worth examining?We thank the reviewer for this comment. We indeed examined this and find that Amot can co-IP with E-cadherin. Moreover, as in the case of PATJ/Pals1 the association is enhanced with the Amot-S176E mutant. We include this new information in 12) The interpretation that the interaction between YAP and Merlin is predominantly occurring in the nucleus and the cytoplasm is supported by the data, but quantification of these experiments would help providing an assessment as to whether this simply reflect the relative levels of YAP across the different fractions or whether there is evidence for a truly differential interaction across fractions. . Perhaps an alternative way to provide support for a compartment-specific interaction would be to employ a fluorescence-based approach .As noted by the reviewer, the data support the formation of the Merlin/YAP/Amot complex predominantly in the cytoplasm and nucleus  and this13) The presentation of the western blots (IP/western) should be accompanied by quantification. I was surprised that experiments that I would assume should be loaded on the same SDS page gel  What is the intracellular localization of endogenous Merlin (and YAP)? The localization experiments currently presented all depend upon transient transfection.The localization of endogenous Merlin and Amot can be seen in the IF experiments shown in 15) For We provide additional support in 16) If the phosphospecific antibody works in IF, it would be nice to provide additional evidence that the phosphorylated form is enriched in the plasma membrane fraction . This would support the results obtained with the S to E mutation.Unfortunately, we were unable to optimize the antibody for IF and therefore unable to carry out this experiment.17) What is the relative level of expression (in relation to the endogenous) of the different Amot constructs? Similarly, the authors may want to comment on the expression levels of the other constructs they used in relation to the respective endogenous proteins. Could some of the results observed due to overexpression / tagging artifacts?Please see our response to question 1.18) Concerning the Please see our response to question 5.19) Other comments: – this likely happened through PDF generation during submission, but the quality of the images is problematic. Labels do not display clearly, and microscopy images are extremely difficult to evaluate. The authors need to provide high quality images with the revision.Thank you for point this out. We redid the images at a higher resolution.


The primary objective was to determine human papilloma virus (HPV) clearance rate after cervical biopsy among women with persistent high-risk HPV infection compared with spontaneous HPV clearance rate in the absence of biopsy.We collected data from a dedicated screening program of women aged 30–70 years old. Inclusion criteria for the baseline non-interventional cohort were a positive HPV test  and normal cytology. In the baseline cohort women were followed with approximately yearly HPV-tests and cytology until HPV regressed (one negative HPV test) or interventions in the form of diagnostic biopsies or therapy. Women who had a diagnostic biopsy were included in the biopsy cohort and followed until HPV regression or therapy. Observed HPV regression rates and time to HPV regression were compared between baseline and biopsy cohorts. For the comparison, we used Fisher’s exact test for the HPV regression rates and interval-censored, accelerated failure time model for time to HPV regression.Among the 1079 women included in the baseline cohort, 499 (46.3%) had HPV clearance and 475 were referred for colposcopy with biopsy. The biopsy cohort comprised all women who were not treated and had at least one HC2 test after biopsy . Of those, 138 (68.7%) experienced HPV regression. In the biopsy cohort, time to clearance of HPV infection was approximately halved  compared with the baseline cohort. This result was robust in a wide range of sensitivity analyses.A higher proportion of women cleared their HPV infection, and time to HPV clearance was shorter in the biopsy cohort than in the baseline cohort. It is reassuring for clinicians to know that conservative management of patients with HPV persistency is successful when colposcopy with biopsies excludes high-grade disease. It is well established that infection of the uterine cervix with high-risk human papillomavirus (HPV) types needs to persist for many years or even decades before invasive cervical cancer develops. There are different approaches to estimate the risk of malignant progression associated with HPV infections and the overall risk of cervical cancer. The life-time risk for cervical cancer reaches 6.5% in some areas without cervical cancer screening . HoweverStudies on the clinical course of HPV infections of the uterine cervix require colposcopy with histological assessment (biopsy) at recruitment because cytology or colposcopic impression alone may under- or overestimate the grade of associated CIN lesions . AlthougLong-term observational studies of women with incident or prevalent HPV infections suggest a typical clearance pattern with a high regression rate in the first year of observation and lower regression rates in subsequent follow-up years . It was Wolfsburg pilot project for better prevention of cervical cancer with Primary HPV Screening (WOLPHSCREEN) has been described in detail elsewhere [The lsewhere . In brielsewhere . AbnormaWomen with positive HC2 tests and normal cytology are followed in yearly intervals. After each year, HC2 testing is repeated and those women with a persistent positive HC2 test may be referred for colposcopy. The colposcopy protocol mandates biopsies for all women with a persistent, positive HC2 test result after 1 year, including those with normal Pap smear test results; therefore, more than 90% of referred women undergo histological assessment.The ethics committee of Lower Saxony in Hannover was consulted but approval and/or trial registration was not required because statutory health insurances Deutsche BKK and Audi BKK use the pilot project as their routine screening program. WOLPHSCREEN is managed by Klinikum Wolfsburg, the central database is located in the department of gynecology. The contract between health insurances, gynecologists in private practice, other partners, and Klinikum Wolfsburg encourages the use of WOLPHSCREEN data for research and gives all rights for analyses and publications to the head of the department of obstetrics and gynecology as leader of the scientific team. Participants need to give written consent to be included in WOLPHSCREEN. The written consent includes an agreement that pseudonymized data and left-over material may be used for research.We followed women aged 30 to 70 years with a positive HC2 test and normal Pap smear at recruitment and at least one further HC2 prior to any intervention or colposcopy to determine the natural course of HPV infection. Women stay in the baseline cohort until regression of HPV infection (the first negative HC2 result), cervical biopsy or therapy, or their last HC2 test, whichever occurs first. Women from the baseline group with a positive HC2 test for at least 1 year can be subsequently referred for colposcopy/biopsy and then included in the ‘biopsy cohort’. In the biopsy cohort, women were observed until HPV regression, further biopsy or therapy, or their last HC2 test (the same criteria as in the baseline cohort). The ‘biopsy cohort’, therefore, comprises a subsample of women from the baseline group at a different time point.First, the distribution of sociodemographic and clinical characteristics was compared for both cohorts by means of tabulation and chi-squared test. In this analysis, we did not exclude women who contributed more than one baseline period . Furthermore, we ignored that women included in the biopsy cohort were a subsample of women in the baseline cohort. In the next step, we used an accelerated failure time (AFT) regression model to compare regression of HPV infection in the biopsy cohort with the baseline cohort. AFT is an alternative approach to Cox regression . In the The effects of changing the inclusion criteria for baseline and/or biopsy cohort were studied in four sensitivity analyses (1) further biopsies were no longer considered a censoring event in the biopsy cohort and were included in the analysis; (2) women with ASC-US or more at the time of inclusion in the biopsy cohort were excluded after biopsy; (3) women developing CIN3 at any point during follow-up were excluded in both cohorts; and (4) only the first follow-up period per woman was included in the analysis.From 29,140 follow-up periods available for 22,029 women, 1079 follow-up periods matched all inclusion criteria for the baseline cohort  showed spontaneous HPV regression without any intervention, 10 (0.9%) underwent immediate excisional therapy, and 95 (8.8%) had persistent HPV infection at the end of follow up, but did not have immediate colposcopy for a variety of reasons . The remaining 475 women were referred for biopsy. Among them 201 (42.3%) had follow-up periods with additional HC2 test results and were included in the ‘biopsy cohort’. Of these, 138 (68.7%) experienced HPV clearance and 53 (26.4%) required further biopsies  148 women were referred for excisional treatment without further HPV tests between biopsy and treatment; b) 38 women had a second biopsy before subsequent HPV testing (in these women HPV infection cleared in 27 cases); these 38 cases were included in the sensitivity analysis ‘multiple biopsies allowed’; c) 43 cases whose last HPV test before transition to the biopsy cohort was positive and their first HPV test after the biopsy was negative; these cases represent spontaneous regression, however, we adopted this conservative approach (reducing the possible effects of biopsies) because HPV testing was not done at the time of biopsy and, therefore, it was not clear if clearance occurred in the baseline cohort or after the transition to the biopsy cohort; d) the remaining 45 women were lost to follow-up, mostly because the time for their next scheduled visit was not reached yet at the time of our analysis.The proportion of women who cleared HPV infection was consistently higher in the biopsy cohort than in the baseline cohort Fig. . In the Overall, the results confirm our a priori hypothesis that taking biopsies increases the clearance rate of high-risk HPV infections. The biopsy cohort comprised women with persistent HPV infection from the start of observation for at least 1 year; despite this persistent infection, we observed a faster regression rate of HPV infections in the biopsy cohort than among the baseline cohort. Our data indicate a direct impact of taking biopsies on the subsequent clinical course of HPV infection. In studies examining the clinical course of prevalent HPV infections, the majority of those reporting high regression rates during the first year of observation referred all or at least a substantial proportion of participants for colposcopy with biopsy at the time of recruitment or shortly thereafter , 11–13. The strengths of this study are the large sample size and its population-based character. Despite the large size, all investigations were conducted by a single team using a standardized protocol. The main limitation is that the data do not come from a clinical trial but from real-life cervical cancer screening. Therefore, the probability of censoring (due to treatment or biopsy) is not independent of the results of the HPV tests. In many cases, it is not clear if this under- or overestimates the effect of biopsy, but in cases where the direction of effect is clear, it results in an underestimation and, therefore, we consider our final estimate of biopsy effects as conservative. Furthermore, women screened in the WOLPHSCREEN program are followed up consistently and systematically according to published protocols . Given tHPV persistency over one or more years identifies women at increased risk for long-term viral persistence and the development of CIN3+ , 14. By The findings were also consistent in the sensitivity analyses. The inclusion of follow-up periods with multiple biopsies, most likely indicating HPV persistence, introduces potential bias because these cases were included only in the biopsy cohort and not in the baseline cohort. Consequently, and as expected, in the sensitivity analyses allowing multiple biopsies, the estimates for the effect of biopsy were smaller (indicative of a bias in a conservative direction), but still significant. The advantage of HPV screening is the detection of CIN3 lesions and cancers (CIN3+) that are missed by cytology. These cases of underlying CIN3+ in women with HPV persistence are assumed to have a very low regression rate. As the risk of CIN3+ was higher in the baseline cohort, we did a sensitivity analysis that excluded all cases of CIN3+ in all cohorts to avoid a possible bias related to the different nature of these cases. However, we still observed an increased regression rate of formerly persisting HPV infections in the biopsy cohort compared with the prevalent HPV infections in the baseline cohort. The finding of faster clearance of persistent HPV infection after biopsy among older women is unexpected and intriguing. Previously, we have shown that HC2-testing with a sensitive relative light unit threshold (RLU 1.0) seems reliable even in older women because of cross-hybridization with some potentially oncogenic HPV subtypes . NeverthOur findings are relevant for clinicians and patients because they suggest that observational management of women with HPV persistency is an effective approach. When high-grade disease is excluded by colposcopy with biopsies, the remaining risk of CIN3 during long-term observation is very low , 16, andWe conclude that all studies of the natural history of HPV face a dilemma. On the one hand, the exact identification of HPV-associated disease depends on high-quality diagnoses during follow-up, which in the case of cervical disease are based on colposcopy; however, the accuracy of colposcopy is determined by an appropriate number of punch biopsies , 18. On


Hepcidin is the master regulator of systemic iron homeostasis. Derived primarily from the liver, it inhibits the iron exporter ferroportin in the gut and spleen, the sites of iron absorption and recycling respectively. Recently, we demonstrated that ferroportin is also found in cardiomyocytes, and that its cardiac-specific deletion leads to fatal cardiac iron overload. Hepcidin is also expressed in cardiomyocytes, where its function remains unknown. To define the function of cardiomyocyte hepcidin, we generated mice with cardiomyocyte-specific deletion of hepcidin, or knock-in of hepcidin-resistant ferroportin. We find that while both models maintain normal systemic iron homeostasis, they nonetheless develop fatal contractile and metabolic dysfunction as a consequence of cardiomyocyte iron deficiency. These findings are the first demonstration of a cell-autonomous role for hepcidin in iron homeostasis. They raise the possibility that such function may also be important in other tissues that express both hepcidin and ferroportin, such as the kidney and the brain.DOI:http://dx.doi.org/10.7554/eLife.19804.001 Many proteins inside cells require iron to work properly, and so this mineral is an essential part of the diets of most mammals. However, because too much iron in the body is also bad for health, mammals possess several proteins whose role is to maintain the balance of iron. Two proteins in particular, called hepcidin and ferroportin, are thought to be important in this process. Some ferroportin is found in the cells that line the gut (where iron is absorbed into the body) and is required to release this iron into the bloodstream. It is also found in the spleen, which is where iron is removed from old red blood cells so that it can be recycled. The liver produces hepcidin to control when ferroportin is active in the gut and spleen.Both hepcidin and ferroportin are also found in heart cells. In 2015, a study reported that that heart ferroportin plays an important role in heart activity. However, it was not clear what role hepcidin plays in this organ.Now, Lakhal-Littleton et al. – including many of the researchers from the previous work – have genetically engineered mice such that they specifically lacked heart hepcidin, or had a version of ferroportin in their heart that does not respond to hepcidin. The experiments show that these changes caused fatal heart failure in the mice because ferroportin releases iron from heart cells in an uncontrolled manner. Lakhal-Littleton et al. were able to prevent heart failure by injecting the animals with iron directly into the bloodstream. These findings show that hepcidin produced outside the liver has a role in controlling the levels of iron in the body’s organs.Other organs such as the brain, kidney and placenta all have their own forms of hepcidin and ferroportin; further work could investigate the roles of these proteins. Finally, another challenge for the future will be to test whether new drugs that are being developed to block or mimic hepcidin from the liver have the potential to treat heart conditions in humans.DOI:http://dx.doi.org/10.7554/eLife.19804.002 Slc40a1), is the only known mammalian iron export protein and mediates iron release into the circulation from duodenal enterocytes, splenic reticuloendothelial macrophages and hepatocytes, the sites of iron absorption, recycling and storage respectively  and divalent metal transporter (DMT1), and the iron storage protein ferritin . Systemiectively . FPN-medectively . The impectively , and in ectively .Other than the liver, hepcidin is also found in tissues with no recognized role in systemic iron homeostasis, including the heart , the braHamp gene, and the second, with cardiomyocyte-specific knock-in of Slc40a1 C326Y, that encodes a FPN with intact iron export function but impaired HAMP binding . Relative to control cardiomyocytes at the respective timepoint, Hamp mRNA was increased by FAC from 4 hr of treatment, and decreased by DFO at 4, 8 and 16 hr of treatment . HAMP prNext, we aimed to understand the mechanisms underlying increased HAMP secretion in DFO-treated cardiomyocytes. To this end, we investigated the role of the prohormone convertase Furin, which in hepatocytes, is required for cleavage of the propeptide and the release of the mature HAMP peptide . Furin eHamp knockout mice fl/fl;Myh6.Cre+Hamp by crossing in-house conditional Hamp floxed (fl) mice with mice transgenic for Cre recombinase under the control of cardiomyocyte-specific promoter Myosin Heavy Chain 6 (Myh6.Cre+). Hamp mRNA  and natriuretic peptide precursor (Nppb)  in controls . Assessmtrophied . This war (Nppb) . TUNEL scontrols .fl/fl;Myh6.Cre+Hamp mice and fl/flHamp littermate controls at 3, 6 and 9 months of age. Mid-ventricular cine MR images showed no differences between the two genotypes at 3 months of age. At 6 and 9 months of age, cine MR images showed marked enlargement of the LV in fl/fl;Myh6.Cre+Hamp mice compared to fl/flHamp controls    and at ee (LVES) , accompae (LVES) . Other pe (LVES) . Taken tMyh6.Cre+ mice have normal cardiac function compared to wild type littermate controls , was absent in iron-treated fl/fl;Myh6.Cre+Hamp mice, consistent with correction of cardiomyocyte iron deficiency  and in the hearts of mice fed an iron-deficient diet  complexes in cardiac lysates from fl/fl;Myh6.Cre+Hamp hearts at 3 months and 6 months of age. We found that Aconitase I, Complex I and Complex IV activities were significantly reduced in fl/fl;Myh6.Cre+Hamp hearts compared to fl/flHamp controls at 3 months and 6 months of age, and that this reduction in activity was prevented in iron-treated 6-month old mice , dilation of mitochondrial cristae was seen in fl/fl;Myh6.Cre+Hamp hearts as early as 3 months of age and progressed further at 6 months of age. However, this was prevented in iron-treated fl/fl;Myh6.Cre+Hamp mice, which had healthy-looking mitochondria at 6 months of age .Having confirmed a causal relationship between cardiomyocyte iron deficiency and cardiac dysfunction, we aimed to understand the mechanisms linking the two. Iron is a cofactor for several enzymes involved in metabolism , and metent diet . Based oold mice . As ETC s of age . SimilarHk2) which catalyses the first step of glycolysis, Enolase (Eno), which catalyses the penultimate step of glycolysis and of Lactate dehydrogenase A (Ldha) which catalyses the ultimate step of glycolysis were all significantly increased at 3 months and 6 months of age in hearts of untreated fl/fl;Myh6.Cre+Hamp mice. This upregulation of glycolytic genes was not seen in iron-treated fl/fl;Myh6.Cre+Hamp mice  is detrimental to cardiac function, it is protective under conditions of increased systemic iron availability (systemic models). Previously, we also showed that deletion of cardiomyocyte FPN resulted in fatal cardiomyocyte iron overload, preventable by dietary iron restriction  ,and alsoAnother open question is whether disruption of the cardiac HAMP/FPN axis contributes to the pathophysiology of heart disease. It has been shown, in the rat model of myocardial infarction (MI), that Hamp mRNA and HAMP protein are elevated in the ischemic myocardium during the acute phase . In humaCurrently, there is considerable interest in targeting the HAMP/FPN axis for the treatment of iron overload and iron deficiency. Our studies suggest that such strategies may also impinge on cardiac iron homeostasis and function. Other than the heart, both FPN and hepcidin are also found in the brain, kidney and placenta . It woulAll animal procedures were compliant with and approved under the UK Home Office Animals (Scientific Procedures) Act 1986. Both males and females were used in experiments, with the respective littermate control being of a matching sex.Hamp allele into C57BL/6N mouse ES cells (JM8F6) with exons 2 and 3, which encode the majority of the peptide, flanked by LoxP sites and a line of floxed mice was generated by blastocyst injection of targeted ES cells, as previous described  promoter (B6.FVB-Tg(Myh6-cre)2182Mds/J).The subsequent breeding strategy was designed to produce cardiac Hamp knockouts and homozygous floxed controls  in the same litter.The strategy for generating cardiac Hamp knockout mice is outlined in escribed . FurtherSlc40a1 C326Y knock-in mice is outlined in The strategy for generating Cardiac 2 and positioned supine in a purpose-built cradle. ECG electrodes were inserted into the forepaws, a respiration loop was taped across the chest and heart and respiration signals were monitored using a custom-built physiological motion gating device. The cradle was lowered into a vertical-bore, 11.7 T MR system with a 40 mm birdcage coil  and visualised using a Bruker console running Paravision 2.1.1. A stack of contiguous 1 mm thick true short-axis ECG and respiration-gated cine-FLASH images were acquired. The entire in vivo imaging protocol was performed in approximately 60 min. Image analysis was performed using ImageJ . Left ventricular volumes and ejection fractions were calculated from the stack of cine images as described , or an iron-loaded diet  or a matched control diet  from weaning for six weeks.Adult primary cardiomyocytes were isolated from eight week old C57BL/6 mice. Hearts were cannulated and mounted on a langendorff apparatus, then perfused using a liberase solution for 10 min. After filtration through a 400 µm gauze, cells were cultured in MEM medium containing Hanks salts, L-glutamine and antibiotics. Within 2 hr of cardiomyocyte culture, supernatants were replaced with fresh medium containing 10% Fetal calf serum, with 0.5 mmol/L ferric citrate (FAC)  or 100 µmol/L desferroxamine  for 8 hr. The Furin inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethylketone (CMK)  was added at a concentration of 50 µmol/L for the duration of DFO and FAC treatment.Slc40a1, Hamp, TfR1, Myh7, Nppb, Ldha, Hk2, Eno and house-keeping gene β-Actin . The CT value for the gene of interest was first normalised by deducting CT value for β-Actin to obtain a delta CT value. Delta CT values of test samples were further normalised to the average of the delta CT values for control samples to obtain delta delta CT values. Relative gene expression levels were then calculated as 2-delta deltaCT.Total RNA extraction and cDNA synthesis were carried out as previously described . Gene exAB_2115844) at 1/40 dilution, or rabbit polyclonal anti-mouse FPN antibody  at 1/200 dilution. Results of control experiments confirming the specificity of the HAMP antibody are shown in Tissues were prepared as described previously  and staiHAMP was measured in mouse sera and in cardiomyocyte supernatants using a HAMP ELISA kit  according to the manufacturer’s instructions. Results of control experiments confirming that in vitro treatments did not affect HAMP peptide detection by this ELISA kit are shown in Formalin-fixed paraffin-embedded tissue sections were deparaffinised using Xylene, then rehydrated in ethanol. Slides were then stained for 1 hr with 1% potassium ferricyanide in 0.1 mol/L HCl buffer. Endogenous peroxidase activity was quenched, then slides were stained with DAB chromogen substrate and counterstained with haematoxylin. They were visualised using a standard brightfield microscope.3 slices were fixed by immersion for 2 hr in 2.5% glutaraldehyde in 0.1 mol/L cacodylate buffer and prepared for electron microscopy by standard methods. Briefly, cells were post-fixed in osmium tetroxide (1% w/v in 0.1 mol/L phosphate buffer), stained with uranyl acetate (2% w/v in distilled water), dehydrated through increasing concentrations of ethanol (70–100%) and acetone and embedded in TAAB resin . Ultrathin sections (50–80 nm) were prepared using a Reichert ultracut S microtome and mounted on 200 mesh nickel grids. Sections were lightly counterstained with lead citrate and uranyl acetate and examined with a Jeol transmission electron microscope .Hearts were dissected and 0.5–1 mmAdult cardiomyocytes were isolated from mice of the desired genotype at 9 weeks of age as described above. Cardiomyocytes were then cultured in 24-well plates at equal densities for 16 hr before the efflux experiment was performed as described . BrieflyFerritin concentration in serum and in liver lysates was determined using the ferritin ELISA kit . Serum iron levels were determined using the ABX-Pentra system . Determination of total elemental iron in the heart was carried out by inductively coupled plasma mass spectrometry (ICP-MS) as described previously . CalibraFollowing cardiac perfusion, hearts were dissected into small pieces in ice-cold Hanks buffer, and subject to collagenase P digestion at 37C for 1 hr . Following lysis of red blood cells, cell suspensions were passed through a 70 µm sieve, before being labelled using cardiomyocyte isolation kit . Separation of cardiomyocyte and non-cardiomyocyte fractions was carried out using MACS magnetic separation system according to the manufacturer’s instructions. Cardiac fractions were lysed immediately for ICP-MS analysis.Approximately 10 mg of frozen, crushed tissues were suspended in 200 µl of ice-cold KME buffer , then homogenized by rupturing with a TissueRuptor  over ice. In a plastic cuvette, the cardiac lysate is mixed with assay buffer and slotted into a spectrophotometer. Details of assay buffers and of reaction procedure for each enzyme are detailed in the Appendix.Values are shown as mean ± standard error of the mean (SEM). Comparison of iron indices, enzyme activities and parameters of cardiac function between groups was performed using Student’s T test. p values < 0.05 were deemed as indicating significant differences between groups. Where significant, exact p values for a figure panel are stated in the corresponding figure legend. No explicit power analysis was performed prior to the experiments to determine sample size, since we had no means to reliably estimate the size and variability of the effects of deleting hepcidin on parameters of cardiac function. For Cine MRI assessment of cardiac function, typically 5–11 animals of each genotype were used, with a matching number of littermate controls. For gene expression, iron quantitation and enzyme activity assays from mouse tissues, typically, 3–6 independent biological replicates and matching littermate controls were analysed. Since significant results were obtained from these set of experiments, no further animals were sacrificed. All 'n' values reported refer to independent biological replicates. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by Naama Barkai (Senior Editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors. The reviewers have opted to remain anonymous.Thank you for submitting your article "An essential cell-autonomous role for hepcidin in cardiac iron homeostasis" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:The reviewers found the manuscript to have merit and potentially to make an important contribution to understanding iron homeostasis at the tissue level. For the first time, the authors tested the conjecture that tissue hepcidin production may contribute to local tissue-level iron regulation.However, a number of important issues were raised by the reviewers including the need to be stricter in the methodology used and its description. In particular, the reviewers were seeking further evidence that the data on hepcidin expression and regulation were robust and that bioactive hepcidin was being detected, with hepcidin KOs as controls. Reviewers were reluctant to accept immunohistochemical staining as quantification of bioactive secreted hepcidin. Furthermore, iron efflux experiments need to be described in more detail. Tissue heterogeneity including the possible presence of macrophages may be an issue to resolve. Any further evidence that can be provided on the regulation of hepcidin would strengthen the paper.I have summarised the major issues below:1) Subsection “Hamp expression and regulation in the heart”, first paragraph and 2) Subsection “Hamp expression and regulation in the heart”, first paragraph and 3) To confirm the equivalence of Hamp KO and ferroportin C326Y the survival curves need to be compared. This is a hard endpoint that has produced a surprise previously . If data exists on the survival of Ferroportin C326Y transgenic mice, it should be presented.4) It would be relatively straightforward for the authors to examine the regulation of cardiac Hamp using hemojuvelin floxed (Hfe2 fl/fl) mice  or other approaches, as regulation of hepatic Hamp expression is well understood. Hfe2 is expressed at high levels in the heart and might be relevant there as it is in the liver. The paper would be much stronger if there was some indication of the mechanism of hepcidin regulation in cardiomyocytes.5) The authors refer to other animal models of cardiac iron deficiency, but they don't really distinguish between those caused by dietary iron deprivation, which would have the confounding existence of iron deficiency anemia, and a heart-specific Tfrc knockout model in which iron deficiency is restricted to the heart . The heart-specific Tfrc knockout was shown to have many characteristics that are similar to those described in this paper. One important difference, however, was in the levels of electron transport chain complexes found in untreated and iron treated control and cardiac Hamp-/- mice . Xu et al. reported decreases in the bands representing iron-containing ETC complexes using the same OXPHOS blotting approach, as would be expected in iron deficient cells. Those representative proteins are labile when the complexes do not form properly. How do the authors explain their unexpected results, particularly considering the decreased complex activity shown in 6) Several experiments were carried out using isolated adult mouse cardiomyocytes. How did the authors insure that the cell populations were not contaminated with other cell types, particularly macrophages, which might confound the results?7) The iron efflux experiments shown in 8) 9) The heart sections in eLife. Your article has been favorably evaluated by Naama Barkai (Senior Editor) and a Reviewing Editor.Thank you for resubmitting your article "An essential cell-autonomous role for hepcidin in cardiac iron homeostasis" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.The reviewers generally did not think that the authors use of tissue immunostaining to quantitate a secreted protein is bullet-proof but accept that the authors have enough additional overlapping evidence to give more confidence that the main conclusions are right. They have put together a strong case for hepcidin and ferroportin interacting in an autocrine/paracrine manner in the heart. The genetic argument from the equivalent pathology and similar survival of cardiomyocyte FpnC326Y (cardiomyocyte hepcidin resistance) and cardiomyocyte hepcidin KO is particularly appealing. The autocrine/paracrine function of hepcidin and ferroportin is an old conjecture but this is the first evidence for it.However, the following is required for acceptance:1) In the rebuttal there is a statement that the reviewers did not agree with. The cardiac-specific Tfr1 KO mice are not embryonic lethal – they live into the second postnatal week. The authors should be aware of this and modify the text if needed.2) One reviewer did not believe that the authors had fully explained the outstanding issues with their OXPHOS blot – they thought it was poor quality with much more animal to animal variability than there should be. If possible, the authors should repeat this blot in order to show more consistency between replicates. Alternatively, the authors should delete this western. […]1) Subsection “Hamp expression and regulation in the heart”, first paragraph and Hepcidin is synthetized as an 84 amino acid prepropeptide, containing an N-terminal ER targeting signal sequence (aa 1-24), a proregion (pro) with a consensus Furin cleavage site (aa 25-59) and a C-terminal bioactive iron-regulatory mature peptide (aa 60-84). The antibody used for immunohistochemical detection of hepcidin in the heart is ab30760 from Abcam, which was raised against the human mature peptide (aa60-84). We now include this information in the Methods section of the revised manuscript.The reviewers’ concerns over the specificity of this antibody are ones that we also had, and consequently we did carry out appropriate control experiments. We now include the results of those control experiments in the revised manuscript. First, we showed that staining with this antibody is absent in the heart of cardiac-specific hamp-1 knockouts , despiteThe reviewer is right to point out that the release of the bioactive hepcidin mature peptide requires prohormone convertases, which may or may not be present in the heart. Furin is the prohormone convertase required for release of hepatic bioactive mature hepcidin peptide. In hepatocytes, it has been shown that Furin inhibition leads to decreased release of the active mature peptide and increased release of the larger inactive propetide, and that both of these protein species are detected by assays targeting the mature peptide . Furin is also highly expressed in the heart , raising the intriguing possibility that it may be involved in the production of the bioactive hepcidin mature peptide from cardiomyocytes. To test this hypothesis, we performed additional experiments using the Furin inhibitor Decanoyl-Arg-Val-Lys-Arg-chloromethylketone trifluoroacetate salt(CMK). These experiments showed that while Furin inhibition under iron replete conditions did not decrease secreted HAMP levels (consistent with the release of the propeptide as reported in hepatocytes) , it nonetheless increased iron export from primary cardiomyocytes. Furthermore, the effect of Furin inhibitor on iron export was only seen in Hamp fl/fl and not in Hamp fl/fl Myh6.Cre+ cardiomyocytes. We include these data in Furthermore, we found that increased HAMP release from DFO-treated cardiomyocytes depended on Furin. These data are added to 2) Subsection “Hamp expression and regulation in the heart”, first paragraph and As suggested by the reviewer, we have performed additional experiments with primary cardiomyocytes from cardiac hamp1-ko cells, and found that levels of HAMP proteins were greatly reduced or undetectable in their supernatants. We include this in The reviewer was concerned that physiological control of hepcidin was lost in cardiomyocytes due to the length of culture. First, both in the original and in the additional experiments performed herein, cardiomyocytes were used within 2 hours of harvest. Additionally, as suggested by the reviewer, we carried out a timecourse of treatment with FAC and DFO, and now present these data in the manuscript (new 3) To confirm the equivalence of Hamp KO and ferroportin C326Y the survival curves need to be compared. This is a hard endpoint that has produced a surprise previously . If data exists on the survival of Ferroportin C326Y transgenic mice, it should be presented.We now provide those data as new 4) It would be relatively straightforward for the authors to examine the regulation of cardiac Hamp using hemojuvelin floxed (Hfe2 fl/fl) mice  or other approaches, as regulation of hepatic Hamp expression is well understood. Hfe2 is expressed at high levels in the heart and might be relevant there as it is in the liver. The paper would be much stronger if there was some indication of the mechanism of hepcidin regulation in cardiomyocytes.HJV is one of many putative regulators of cardiac hepcidin. As it happens, muscle-specific HJV KO mice have increased rather than decreased cardiac hepcidin expression . Thus, it appears that both with respect to HJV and to iron, cardiac hepcidin is regulated differently from liver hepcidin. Substantive additional studies are required to explore the role of known regulators of hepatic hepcidin in the regulation of cardiac hepcidin. We consider such studies beyond the scope of this revision as the work would constitute an independent manuscript.5) The authors refer to other animal models of cardiac iron deficiency, but they don't really distinguish between those caused by dietary iron deprivation, which would have the confounding existence of iron deficiency anemia, and a heart-specific Tfrc knockout model in which iron deficiency is restricted to the heart . The heart-specific Tfrc knockout was shown to have many characteristics that are similar to those described in this paper. One important difference, however, was in the levels of electron transport chain complexes found in untreated and iron treated control and cardiac Hamp-/- mice . Xu et al. reported decreases in the bands representing iron-containing ETC complexes using the same OXPHOS blotting approach, as would be expected in iron deficient cells. Those representative proteins are labile when the complexes do not form properly. How do the authors explain their unexpected results, particularly considering the decreased complex activity shown in With regards to the description of animal models of cardiac iron deficiency, we have now amended the text to distinguish between the various models, as suggested by the reviewer. With regards to the levels of ETC complexes detected by OXPHOS, we too were initially surprised that we did not detect a reduction as was seen in cardiac TfR knockout mice .However, this becomes less surprising when you consider the relative severity of cardiac iron deficiency, which is reflected in the fact that cardiac-specific TfR KO are embryonic lethal whereas cardiac-specific hepcidin knockouts have a mean survival of 28 weeks. In this context, it is important to consider the mechanisms through which iron deficiency impinges on the ETC. These mechanisms include not only limiting iron availability for complex assembly and for enzymatic activity of assembled complexes, but also reducing the levels of mitochondrially-encoded proteins. Indeed, the degree of iron deficiency (and iron excess) have been shown to correlate with damage to mitochondrial DNA , and many of the proteins in ETC complexes are encoded by mitochondrial genes. Thus, depending on the degree of iron deficiency in the cardiomyocytes, different mechanisms may come into play, meaning that complex assembly or activity could still be disrupted when levels of individual proteins in the complex are still maintained.6) Several experiments were carried out using isolated adult mouse cardiomyocytes. How did the authors insure that the cell populations were not contaminated with other cell types, particularly macrophages, which might confound the results?The reviewer is right to point out that macrophages are known to be present in the cardiac tissue. We cannot exclude the presence of macrophages in our cardiomyocyte culture. However, specifically with respect to hepcidin production in the supernatants, we can be confident that cardiomyocytes are the source of the hepcidin, because very little hepcidin was detected in supernatants of cardiac-specific hepcidin knockouts (where cardiomyocytes but not macrophages have deletion of the hepcidin gene). We include these data in 7) The iron efflux experiments shown in The method for measuring iron efflux used in our experiments is not novel, but adapted from the method used by Mckie et al. 2000, which was the first publication to describe the function of FPN in iron efflux. We now include this reference in the text. This method does not enable measurement of absolute amounts of iron flux, but rather measurement of iron efflux as a percentage of the total iron taken up during incubation with the uptake solution; percentage efflux= 100 x Fe55 in efflux solution /(Fe55 in efflux+ Fe55 remaining in cells). The readouts are obtained using a scinitillation counter and are expressed as counts per minute. Count rate does not universally equate to dose rate, and there is no simple universal conversion factor. Any conversions are instrument-specific.The reviewer also questions the use of non-transferrin bound Fe55  instead of transferrin-bound Fe55 for loading cells with iron. The rationale for this is to circumvent any confounding effects of differential iron uptake between cells of different genotypes due to differing levels of TfR1 expression. Indeed, we know that TfR1 expression is higher in cardiac-hepcidin knockout cells and in Slc40a1 C326Y knock-in cells than in their respective controls . While TTo minimise carryover of Fe55, cells were washed with PBS 3 times before addition of efflux medium. Efflux measurements were carried out in three independent experiments, with each experiment containing three biological replicates (3 wells of cardiomyocytes) for each genotype and condition. The range between values obtained from three biological replicates constituted between 0.07and 0.21 of the value of mean. Thus, the results were sufficiently accurate to make meaningful comparisons between genotypes and treatment conditions within each experiment, and the outcome of those comparisons was reproducible between experiments.The reviewer raises an interesting question relating to the levels of iron export being higher in Slc40a1 C326Y knock-in cardiomyocytes than in Hamp knockout cardiomyocytes (p=0.014). We too were initially surprised at this result. One possible explanation is that FPN levels in Slc40a1 C326Y knock-in cardiomyocytes are higher than in Hamp KOs cardiomyocytes, due to the fact that FPN in the former setting is completely insensitive to any hepcidin, while FPN in the latter may still be subject to some regulation by hepcidin from non-cardiomyocyte sources.8) -δ δ CT. The reviewer’s questions relating to the discrepancy between hepcidin transcript and protein, the possible role of protein processing and the measurement of hepcidin in supernatants are addressed in responses to points 1 and 2 above.In 9) The heart sections in 4 (range between 45 and 60 um2) in control animals may be due to any number of reasons including: a) the manual nature of quantitation of cardiomyocyte size, which requires drawing around individual WGA-stained cardiomyocytes, which is likely to vary between individual users, b) the method for tissue fixing and processing of the cardiac tissue, c)the area of the heart selected for histology and d) orientation of section plane relative to cardiomyocyte axis.We provide better heart sections in Figure 2bB In terms of cardiomyocyte size, differences in reported cardiomyocyte size between our data (range between 25 and 37 um2) and those of reported by Xu et alThe reviewers generally did not think that the authors use of tissue immunostaining to quantitate a secreted protein is bullet-proof but accept that the authors have enough additional overlapping evidence to give more confidence that the main conclusions are right. They have put together a strong case for hepcidin and ferroportin interacting in an autocrine/paracrine manner in the heart. The genetic argument from the equivalent pathology and similar survival of cardiomyocyte FpnC326Y (cardiomyocyte hepcidin resistance) and cardiomyocyte hepcidin KO is particularly appealing. The autocrine/paracrine function of hepcidin and ferroportin is an old conjecture but this is the first evidence for it.However, the following is required for acceptance:1) In the rebuttal there is a statement that the reviewers did not agree with. The cardiac-specific Tfr1 KO mice are not embryonic lethal – they live into the second postnatal week. The authors should be aware of this and modify the text if needed.We thank the reviewers for drawing our attention to the erroneous statement that “cardiacTfR1 knockout are embryonic lethal”. In the original response letter, the phenotype of these mice was discussed in relation to our results showing the levels of ETC complexes as detected by the OXPHOS assay . As these data have now been removed from the manuscript, so has the erroneous statement in the revised response letter.2) One reviewer did not believe that the authors had fully explained the outstanding issues with their OXPHOS blot – they thought it was poor quality with much more animal to animal variability than there should be. If possible, the authors should repeat this blot in order to show more consistency between replicates. Alternatively, the authors should delete this western.In relation to the OXPHOS blot showing the levels of ETC complexes , we have now run the blot a second time. However, the variability between some of the samples persists. As such, we have opted to remove the blot from the manuscript, and have also removed all text relating to it in the Results, Discussion, Methods and figure legends section. Accordingly, we have also removed any text in our original response letter relating to this particular dataset.


Epidemiologic studies have been difficult to perform due to the lack of International Classification of Diseases (ICD) codes differentiating smoldering from active MM.Smoldering multiple myeloma (SMM) is currently defined as MM without evidence of impending  or active (hyperN=92 993). Follow-up data were collected until the end of 2012. NCDB records the time to initial treatment or reasons for not receiving treatment. We considered a patient to have active MM if any treatment (chemotherapy and/or radiation) was recommended by the attending physician within 120 days of MM diagnosis regardless of whether it was actually administered. Reasons for not administering treatment included co-morbidities, advanced age, patient refusal or death before treatment could be initiated. Patients who did not require treatment within 120 days of MM diagnosis were considered to have SMM. Patients with the following characteristics (due to missing data or short follow-up) were considered to have MM with unknown disease activity: (a) vital status alive or unknown and treatment not recommended but follow-up <120 days; and (b) treatment recommendation not recorded and vital status alive or unknown but with ⩾120 days of follow-up. Since the cause of death is not captured in NCDB, we considered patients who died within 120 days of MM diagnosis to have active MM even if no treatment was initially recommended as we could not exclude transformation into active MM. A detailed algorithm of disease activity classification is shown in n=157). Patients who were diagnosed (n=6473) but did not receive any treatment at the reporting facility (class of case 00) were excluded from the survival analysis as follow-up information might be incomplete. We analyzed the Surveillance, Epidemiology, and End Results Program (SEER) data from 2003–2011 (18 registries) using SEER*Stat software version 8.3.2  to determine the incidence of all MM age-adjusted to the 2000 US standard population. The incidence of SMM was then derived from this based on the proportion of MM considered to be smoldering. SEER collects cancer incidence and survival data from cancer registries covering ~28% of the US population (www.seer.cancer.gov).Utilizing the National Cancer Data Base (NCDB), we estimated the overall proportion of new adult MM (ICD-O: 9732) cases that were smoldering and analyzed the data according to socio-geo-demographic subgroups and type of treatment facility. We also compared the overall survival (OS) of SMM to active MM. NCDB, a joint program of the Commission on Cancer of the American College of Surgeons and the American Cancer Society, is the largest public cancer database in the US. It receives oncology outcomes data from ~1500 Commission on Cancer-accredited cancer programs covering >70% of all newly diagnosed cancer cases in the US. We included all new MM patients diagnosed from 2003 to 2011 . The proportions of SMM according to socio-geo-demographic subgroups as well as co-morbidity and type of treatment facility are shown in P=0.27). A recent population-based study showed that Blacks had a longer MM-specific survival compared with Whites.5 This was despite the fact that Blacks were less likely to receive high-dose chemotherapy and autologous stem cell transplantation in the up-front setting.6 The higher prevalence of SMM among Blacks may in part explain this paradox. This is in addition to the recent finding that Blacks may have a higher prevalence of more favorable cytogenetic abnormalities.7 Because our study included an unselected population of MM patients, the OS for active MM was lower compared with previous institutional data from the same era,8 although similar to SEER (data not shown). Population or institutional studies reporting OS for smoldering MM that included those who did and did not progress into active MM are not available for comparison. However, we expect patients with smoldering MM to have a lower OS compared with the general population even if they did not progress into active MM. This is because they would have co-morbidities that presented as signs or symptoms mimicking MM.Of the 86 327 MM patients included in the study, 13.7% were SMM with a median age at diagnosis of 67 years. The estimated incidence was 0.9 cases per 100 000 persons. These findings are similar to a recent Swedish population-based study, wherein 14.4% of MMs were smoldering at diagnosis with an incidence of 0.4 cases per 100 000 persons. However, the algorithm for classifying MM as active versus smoldering was not described.9 This current study included only MM patients diagnosed until the year 2011, three years before the International Myeloma Working Group updated the criteria defining disease activity.1 We estimate that ~10–15% of SMM in our study population would be upstaged to active MM using these new criteria.2Our study included a large cohort comprised of >10 000 SMM patients with varied sociodemographic and geographic subgroups that is likely representative of the US population. However, we do acknowledge certain limitations. In 5.7% of the MM patients, there were missing data pertaining to treatment recommendation or follow-up status. Therefore, we were unable to definitely classify this subgroup of patients as active or smoldering. Nevertheless, it is reassuring that our current estimate is very similar to what was found in a recent study of the SEER-Medicare population (15.2%), in which there was not only access to the type of disease complications at the time of MM diagnosis but also the specific treatment received.Approximately one in seven patients with MM in the US is smoldering at diagnosis. The prevalence, but not the OS, of SMM varies among various sociodemographic and geographic subgroups. Epidemiologic studies in MM should take into account an estimate of those with smoldering disease when studying population disparities in treatment utilization and survival outcome. Our results can be used in the future to study the health care impact of SMM in the US.