Endothelial ZIP8 plays a minor role in BMP6 regulation by iron in mice.

ABSTRACT: Iron-mediated induction of bone morphogenetic protein (BMP)6 expression by liver endothelial cells is essential for iron homeostasis regulation. We used multiple dietary and genetic mouse cohorts to demonstrate a minor functional role for the metal-ion transporter ZIP8 in regulating BMP6 expression under high-iron conditions.

Bone morphogenetic protein 6 (BMP6) antagonises experimental proliferative vitreoretinopathy established by TGF-ß2 stimulation in retinal pigment epithelial cells through modulation of the p38 and JNK MAPK pathways.

The formation of the epiretinal fibrotic membrane by retinal pigment epithelial (RPE) cells is a primary pathological change for proliferative vitreoretinopathy (PVR). Bone morphogenetic protein 6 (BMP6) is an antifibrogenic factor in various cells. To date, it is still unknown whether BMP6 can interfere with the fibrogenesis of RPE cells during the progression of PVR. This work aimed to address the relationship between BMP6 and transforming growth factor-ß2 (TGF-ß2)-elicited fibrogenesis of RPE cells, an experimental model for studying PVR in vitro. The BMP6 level was down-regulated, while the TGF-ß2 level was up-regulated in the vitreous humor of PVR patients. The BMP6 level was down-regulated in human RPE cells challenged with TGF-ß2. The treatment of RPE cells with TGF-ß2 resulted in significant increases in proliferation, migration, epithelial-to-mesenchymal transition (EMT), and extracellular matrix (ECM) remodelling. These effects were found to be inhibited by the overexpression of BMP6 or exacerbated by the knockdown of BMP6. BMP6 overexpression reduced the phosphorylation of p38 and JNK in TGF-ß2-stimulated RPE cells, while BMP6 knockdown showed the opposite effects. The inhibition of p38 or JNK partially reversed the BMP6-silencing-induced promoting effects on TGF-ß2-elicited fibrogenesis in RPE cells. Taken together, BMP6 demonstrates the ability to counteract the proliferation, migration, EMT, and ECM remodelling of RPE cells induced by TGF-ß2. This is achieved through the regulation of the p38 and JNK MAPK pathways. These findings imply a potential connection between BMP6 and PVR, and highlight the potential application of BMP6 in therapeutic interventions for PVR.

miR-206a-3p suppresses the proliferation and differentiation of chicken chondrocytes in tibial dyschondroplasia by targeting BMP6.

The poultry skeletal system serves multiple functions, not only providing structural integrity but also maintaining the balance of essential minerals such as calcium and phosphorus. However, in recent years, the consideration of skeletal traits has been overlooked in the selective breeding of broilers, resulting in an inadequate adaptation of the skeletal system to cope with the rapid increase in body weight. Consequently, this leads to lameness and bone diseases such as tibial dyschondroplasia (TD), which significantly impact the production performance of broilers. Accumulating evidence has shown that microRNAs (miRNA) play a crucial role in the differentiation, formation, and disease of cartilage. However, the miRNA-mediated molecular mechanism underlying chicken TD formation is still poorly understood. The objective of this study was to investigate the biological function and regulatory mechanism of miRNA in chicken TD formation. Based on transcriptome sequencing of tibial cartilage in the healthy group and TD group, miR-206a-3p was found to be highly expressed in TD cartilage. The function of miR-206a-3p was explored through the transfection test of miR-206a-3p mimics and miR-206a-3p inhibitor. In this study, we utilized qRT-PCR, CCK-8, EdU, western blot, and flow cytometry to detect the proliferation, differentiation, and apoptosis of chondrocytes. The results revealed that miR-206a-3p suppressed the proliferation and differentiation of TD chondrocytes while promoting their programmed cell death. Furthermore, through biosynthesis and dual luciferase assays, it was determined that BMP6 was the direct target gene of miR-206a-3p. This finding was further supported by rescue experiments which confirmed the involvement of BMP6 in the regulatory pathway governed by miR-206a-3p. Our results suggest that miR-206a-3p can inhibits the proliferation and differentiation promote apoptosis through the target gene BMP-6 and suppressing the Smad2/3 signaling pathway in chicken TD chondrocytes.

The hepatokine FGL1 regulates hepcidin and iron metabolism during anemia in mice by antagonizing BMP signaling.

ABSTRACT: As a functional component of erythrocyte hemoglobin, iron is essential for oxygen delivery to all tissues in the body. The liver-derived peptide hepcidin is the master regulator of iron homeostasis. During anemia, the erythroid hormone erythroferrone regulates hepcidin synthesis to ensure the adequate supply of iron to the bone marrow for red blood cell production. However, mounting evidence suggested that another factor may exert a similar function. We identified the hepatokine fibrinogen-like 1 (FGL1) as a previously undescribed suppressor of hepcidin that is induced in the liver in response to hypoxia during the recovery from anemia, and in thalassemic mice. We demonstrated that FGL1 is a potent suppressor of hepcidin in vitro and in vivo. Deletion of Fgl1 in mice results in higher hepcidin levels at baseline and after bleeding. FGL1 exerts its activity by directly binding to bone morphogenetic protein 6 (BMP6), thereby inhibiting the canonical BMP-SMAD signaling cascade that controls hepcidin transcription.

Bone morphogenetic protein 6 induces downregulation of pentraxin 3 expression in human granulosa lutein cells in women with polycystic ovary syndrome.

PURPOSE: To evaluate whether PTX3 is differentially expressed in the granulosa lutein cells derived from women with PCOS and whether BMP6 can regulate the expression of PTX3 in hGL cells. METHODS: The expression levels of BMP6 and PTX3 in granulosa lutein cells were evaluated by RT-qPCR. The correlation between the expression levels of BMP6 /PTX3 and oocyte quality indexes were analyzed using clinical samples. The cells were incubated with BMP6 at different concentrations and times to check the expression of PTX3 in KGN cells. TGF-ß type I inhibitors and small interfering RNA targeting ALK2/3/6,SMAD1/5/8 and SMAD4 were used to study the involvement of SMAD dependent pathways in KGN cells. RESULTS: The levels of BMP6 in hGL cells were negatively correlated with the corresponding oocyte maturation rate and high-quality embryo rate, whereas the levels of PTX3 were positively correlated with the corresponding oocyte maturation rate in PCOS. Additionally, the in vitro cell cultured results showed BMP6 significantly inhibited the expression of PTX3 in KGN cells. Furthermore, using a dual inhibition approach (kinase inhibitors and small interfering RNAs), we identified the ALK2/ALK3 type I receptors and BMPR2/ACVR2A type II receptors and the downstream SMAD1/SMAD5-SMAD4 signaling pathway were responsible for the BMP6-induced cellular activities in KGN cells. CONCLUSIONS: The suppressive effect of BMP6 on PTX3 was mediated by ALK2/ALK3 type I receptors and BMPR2/ACVR2A type II receptors in granulosa cells through the SMAD1/5-SMAD4 dependent signaling pathway in PCOS.Our findings provides new insights into the understanding of the pathogenesis of PCOS-related ovulatory disorders.

An H2 S-BMP6 Dual-Loading System with Regulating Yap/Taz and Jun Pathway for Synergistic Critical Limb Ischemia Salvaging Therapy.

Critical limb ischemia, the final course of peripheral artery disease, is characterized by an insufficient supply of blood flow and excessive oxidative stress. H2 S molecular therapy possesses huge potential for accelerating revascularization and scavenging intracellular reactive oxygen species (ROS). Moreover, it is found that BMP6 is the most significantly up-expressed secreted protein-related gene in HUVECs treated with GYY4137, a H2 S donor, based on the transcriptome analysis. Herein, a UIO-66-NH2 @GYY4137@BMP6 co-delivery nanoplatform to strengthen the therapeutic effects of limb ischemia is developed. The established UIO-66-NH2 @GYY4137@BMP6 nanoplatform exerts its proangiogenic and anti-oxidation functions by regulating key pathways. The underlying molecular mechanisms of UIO-66-NH2 @GYY4137@BMP6 dual-loading system lie in the upregulation of phosphorylated YAP/TAZ and Jun to promote HUVECs proliferation and downregulation of phosphorylated p53/p21 to scavenge excessive ROS. Meanwhile, laser-doppler perfusion imaging (LDPI), injury severity evaluation, and histological analysis confirm the excellent therapeutic effects of UIO-66-NH2 @GYY4137@BMP6 in vivo. This work may shed light on the treatment of critical limb ischemia by regulating YAP, Jun, and p53 signaling pathways based on gas-protein synergistic therapy.

BMP5 contributes to hepcidin regulation and systemic iron homeostasis in mice.

Hepcidin is the master regulator of systemic iron homeostasis. The bone morphogenetic protein (BMP) signaling pathway is a critical regulator of hepcidin expression in response to iron and erythropoietic drive. Although endothelial-derived BMP6 and BMP2 ligands have key functional roles as endogenous hepcidin regulators, both iron and erythropoietic drives still regulate hepcidin in mice lacking either or both ligands. Here, we used mice with an inactivating Bmp5 mutation (Bmp5se), either alone or together with a global or endothelial Bmp6 knockout, to investigate the functional role of BMP5 in hepcidin and systemic iron homeostasis regulation. We showed that Bmp5se-mutant mice exhibit hepcidin deficiency at age 10 days, blunted hepcidin induction in response to oral iron gavage, and mild liver iron loading when fed on a low- or high-iron diet. Loss of 1 or 2 functional Bmp5 alleles also leads to increased iron loading in Bmp6-heterozygous mice and more profound hemochromatosis in global or endothelial Bmp6-knockout mice. Moreover, double Bmp5- and Bmp6-mutant mice fail to induce hepcidin in response to long-term dietary iron loading. Finally, erythroferrone binds directly to BMP5 and inhibits BMP5 induction of hepcidin in vitro. Although erythropoietin suppresses hepcidin in Bmp5se-mutant mice, it fails to suppress hepcidin in double Bmp5- and Bmp6-mutant males. Together, these data demonstrate that BMP5 plays a functional role in hepcidin and iron homeostasis regulation, particularly under conditions in which BMP6 is limited.

CREB-H is a stress-regulator of hepcidin gene expression during early postnatal development.

Hepcidin, the hepatic iron hormone, is the central regulator of iron homeostasis. Cyclic AMP-Responsive Element-Binding protein 3-like 3 (CREB3L3/CREB-H) is a liver homeostatic regulator of essential nutrients (i.e. glucose and lipids) and has been previously involved in hepcidin response to pathologic stress signals. Here, we asked whether CREB-H has also a physiologic role in iron homeostasis through hepcidin. To this end, we analyzed hepcidin gene expression and regulation in the liver of wild type and Creb3l3 knockout mice during early postnatal development, as a model of "physiologic" stressful condition. The effect of iron challenge in vivo and BMP6 stimulation in vitro have been also addressed. In addition, we investigated the BMP signaling pathway and hepcidin promoter activity following CREB3L3 silencing and hepcidin promoter mutation in HepG2 cells. Creb3l3 knockout suckling and young-adult mice showed a prominent serum and hepatic iron accumulation, respectively, due to impaired hepcidin mRNA expression which progressively returned to normal level in adult mice. Interestingly, upon iron challenge, while the upstream BMP/SMAD signaling pathway controlling hepcidin was equally responsive in both strains, hepcidin gene expression was impaired in knockout mice and more iron accumulated in the liver. Accordingly, hepcidin gene response to BMP6 was blunted in primary CREB-H knockout hepatocytes and in HepG2 cells transfected with CREB-H siRNA or carrying a hepcidin promoter mutated in the CREB-H binding site. In conclusion, CREB-H has a role in maintaining the homeostatic balance of iron traffic through hepcidin during the critical postnatal period and in response to iron challenge. KEY MESSAGES: CREB-H KO mice develop liver iron overload shortly after weaning that normalizes in adulthood. CHEB-H is involved in hepcidin gene response to oral iron in vivo. CREB-H loss hampers hepcidin promoter response to BMP6. CREB-H is a key stress-sensor controlling hepcidin gene transcription in physiologic and pathophysiologic states.

Dynamic chromatin accessibility during nutritional iron overload reveals a BMP6-independent induction of cell cycle genes.

Iron is essential to organism physiology as it participates in numerous biological processes including oxygen transport, respiration, and erythropoiesis. Although iron is critical to physiology, excess iron is toxic to cells and tissues due to generation of reactive oxygen species. Therefore, well-kept iron homeostasis is a mainstay of proper cell and organ function. Iron overload disorders, caused by nutritional or genetic factors, contribute to many pathologies such as diabetes, non-alcoholic steatohepatitis and hepatocellular carcinoma. The liver is not only vulnerable to the effects of iron overload, it is also the major organ controlling iron homeostasis. During iron overload, Bone Morphogenic Protein (BMP) levels increase and initiate a hepatic response aimed at lowering iron levels. The transcriptional effects of iron overload are not well-characterized and the underlining enhancer regulation is uncharted. Here, we profiled the liver's transcriptome and chromatin accessibility following nutritional iron overload. We found marked changes in gene expression and enhancer accessibility following iron overload. Surprisingly, 16% of genes induced following iron overload participate in propagating the cell cycle. Induction of cell cycle genes was independent of BMP. Genome-wide enhancer landscape profiling revealed hundreds of enhancers with altered activity following iron overload. Characterization of transcription factor motifs and footprints in iron-regulated enhancers showed a role for the Activator Protein 1 (AP-1) transcription factor in promoting cell cycle-related transcription. In summary, we found that the transcriptional program at play during iron overload is bifurcated in which BMP signaling controls iron homeostasis genes while an AP-1-driven program controls cell cycle genes.

BMP6 regulates AMH expression via SMAD1/5/8 in goat ovarian granulosa cells.

Anti-Müllerian hormone (AMH) is produced by ovarian granulosa cells (GCs)and plays a major role in inhibiting the recruitment of primordial follicles and reducing the sensitivity of growing follicles to follicle-stimulating hormone (FSH). Bone morphogenetic protein 6 (BMP6) has similar spatiotemporal expression to AMH during follicular development, suggesting that BMP6 may regulate AMH expression. However, the specific mechanism by which BMP6 regulates AMH expression remains unclear. The objectives of this study were to examine the molecular pathway by which BMP6 regulates AMH expression. The results showed that BMP6 promoted the secretion and expression of AMH in goat ovarian GCs. Mechanistically, BMP6 upregulated the expression of sex-determining region Y-box 9 (SOX9) and GATA-binding factor 4 (GATA4), which was associated with the transcriptional initiation of AMH. AMH expression was significantly decreased by GATA4 knockdown. Moreover, BMP6 treatment promoted the phosphorylation of SMAD1/5/8, whereas inhibiting the SMAD1/5/8 signaling pathway significantly abolished BMP6-induced upregulation of AMH and GATA4 expression. Interestingly, the activation of SMAD1/5/8 alone did not affect the expression of AMH or GATA4. The results suggested that BMP6 upregulated GATA4 through the SMAD1/5/8 signaling pathway, which in turn promoted AMH expression.

Anticeramide Improves Sjögren's Syndrome by Blocking BMP6-Induced Th1.

T-cell dysfunction has been shown to play an important role in the pathogenesis of Sjögren's syndrome (SS). In recent studies, the increased expression of BMP6 has been reported to be related to SS. However, the roles that BMP6 plays in immune homeostasis in the development of SS as well as the downstream signals activated by BMP6 remain unclear. In this study, we investigated the effects and molecular mechanisms of BMP6 on naive CD4+ T cells, showing that BMP6 could upregulate interferon (IFN)-γ secretion from CD4+ T cells through a ceramide/nuclear factor-κB pathway, with no effect on T-cell activation or proliferation. Moreover, an in vivo study showed that anticeramide treatment (myriocin) for an SS animal model (NOD/LtJ mice) could significantly decrease the IFN-γ expression and Th1 frequency in the salivary glands and suppress the inflammation infiltration in salivary glands and maintain the salivary flow rates, both of which reflect SS-like symptoms. This study identifies a promising target that could effectively attenuate the abnormal state of CD4+ T cells and reverse the progression of SS.

Liver sinusoidal endothelial cells induce BMP6 expression in response to non-transferrin-bound iron.

Homeostatic adaptation to systemic iron overload involves transcriptional induction of bone morphogenetic protein 6 (BMP6) in liver sinusoidal endothelial cells (LSECs). BMP6 is then secreted to activate signaling of the iron hormone hepcidin (HAMP) in neighboring hepatocytes. To explore the mechanism of iron sensing by LSECs, we generated TfrcTek-Cre mice with endothelial cell-specific ablation of transferrin receptor 1 (Tfr1). We also used control Tfrcfl/fl mice to characterize the LSEC-specific molecular responses to iron using single-cell transcriptomics. TfrcTek-Cre animals tended to have modestly increased liver iron content (LIC) compared with Tfrcfl/fl controls but expressed physiological Bmp6 and Hamp messenger RNA (mRNA). Despite a transient inability to upregulate Bmp6, they eventually respond to iron challenges with Bmp6 and Hamp induction, yet occasionally to levels slightly lower relative to LIC. High dietary iron intake triggered the accumulation of serum nontransferrin bound iron (NTBI), which significantly correlated with liver Bmp6 and Hamp mRNA levels and elicited more profound alterations in the LSEC transcriptome than holo-transferrin injection. This culminated in the robust induction of Bmp6 and other nuclear factor erythroid 2-related factor 2 (Nrf2) target genes, as well as Myc target genes involved in ribosomal biogenesis and protein synthesis. LSECs and midzonal hepatocytes were the most responsive liver cells to iron challenges and exhibited the highest expression of Bmp6 and Hamp mRNAs, respectively. Our data suggest that during systemic iron overload, LSECs internalize NTBI, which promotes oxidative stress and thereby transcriptionally induces Bmp6 via Nrf2. Tfr1 appears to contribute to iron sensing by LSECs, mostly under low iron conditions.

RNF4~RGMb~BMP6 axis required for osteogenic differentiation and cancer cell survival.

Molecular understanding of osteogenic differentiation (OD) of human bone marrow-derived mesenchymal stem cells (hBMSCs) is important for regenerative medicine and has direct implications for cancer. We report that the RNF4 ubiquitin ligase is essential for OD of hBMSCs, and that RNF4-deficient hBMSCs remain as stalled progenitors. Remarkably, incubation of RNF4-deficient hBMSCs in conditioned media of differentiating hBMSCs restored OD. Transcriptional analysis of RNF4-dependent gene signatures identified two secreted factors that act downstream of RNF4 promoting OD: (1) BMP6 and (2) the BMP6 co-receptor, RGMb (Dragon). Indeed, knockdown of either RGMb or BMP6 in hBMSCs halted OD, while only the combined co-addition of purified RGMb and BMP6 proteins to RNF4-deficient hBMSCs fully restored OD. Moreover, we found that the RNF4-RGMb-BMP6 axis is essential for survival and tumorigenicity of osteosarcoma and therapy-resistant melanoma cells. Importantly, patient-derived sarcomas such as osteosarcoma, Ewing sarcoma, liposarcomas, and leiomyosarcomas exhibit high levels of RNF4 and BMP6, which are associated with reduced patient survival. Overall, we discovered that the RNF4~BMP6~RGMb axis is required for both OD and tumorigenesis.

Functional role of endothelial transferrin receptor 1 in iron sensing and homeostasis.

Systemic iron homeostasis is regulated by the hepatic hormone hepcidin to balance meeting iron requirements while limiting toxicity from iron excess. Iron-mediated induction of bone morphogenetic protein (BMP) 6 is a central mechanism for regulating hepcidin production. Liver endothelial cells (LECs) are the main source of endogenous BMP6, but how they sense iron to modulate BMP6 transcription and thereby hepcidin is uncertain. Here, we investigate the role of endothelial cell transferrin receptor 1 (TFR1) in iron uptake, BMP6 regulation, and systemic iron homeostasis using primary LEC cultures and endothelial Tfrc (encoding TFR1) knockout mice. We show that intracellular iron regulates Bmp6 expression in a cell-autonomous manner, and TFR1 mediates iron uptake and Bmp6 expression by holo-transferrin in primary LEC cultures. In addition, endothelial Tfrc knockout mice exhibit altered iron homeostasis compared with littermate controls when fed a limited iron diet, as evidenced by increased liver iron and inappropriately low Bmp6 and hepcidin expression relative to liver iron. However, endothelial Tfrc knockout mice have a similar iron phenotype compared to littermate controls when fed an iron-rich standard diet. Finally, ferritin and non-transferrin bound iron (NTBI) are additional sources of iron that mediate Bmp6 induction in primary LEC cultures via TFR1-independent mechanisms. Together, our data demonstrate a minor functional role for endothelial cell TFR1 in iron uptake, BMP6 regulation, and hepatocyte hepcidin regulation under iron limiting conditions, and suggest that ferritin and/or NTBI uptake by other transporters have a dominant role when iron availability is high.

Foxp1 and Foxp4 Deletion Causes the Loss of Follicle Stem Cell Niche and Cyclic Hair Shedding by Inducing Inner Bulge Cell Apoptosis.

Quiescent hair follicle stem cells (HFSCs) reside in specialized bulge niche where they undergo activation and differentiation upon sensing niche-dependent signals during hair follicle (HF) homeostasis and wound repair. The underlying mechanism of HFSCs and bulge niche maintenance is poorly understood. Our previous study has reported that a transcription factor, forkhead box P1 (Foxp1), functions to maintain the quiescence of HFSCs. Here, we further discovered that forkhead box P4 (Foxp4), a close family member of Foxp1, had similar expression profiles in various components of HFs and formed a complex with Foxp1 in vitro and in vivo. The HF-specific deficiency of Foxp4 resulted in the precocious activation of HFSCs during hair cycles. In contrast to single Foxp1 or Foxp4 conditional knockout (cKO) mice, Foxp1/4 double cKO exerted an additive effect in the spectrum and severity of phenotypes in HFSC activation, hair cycling acceleration and hair loss, coupled with remarkable downregulation of fibroblast growth factor 18 (Fgf18) and bone morphogenetic protein 6 (Bmp6) expression in bulge cells. In addition, the double KO of Foxp1/4 induced the apoptosis of K6-positive (K6+) inner bulge cells, a well-established stem cell (SC) niche, thus resulting in the destruction of the bulge SC niche and recurrent hair loss. Our investigation reveals the synergistic role of Foxp1/4 in sustaining K6+ niche cells for the quiescence of HFSCs.

Regulatory role of dihydrotestosterone on BMP-6 receptors in granular cells of sheep antral follicles.

Bone morphogenetic protein-6 (BMP-6) and dihydrotestosterone (DHT) affect steroid synthesis in follicles and regulate cell proliferation in the ovaries of female animals. However, little is known about granular cells (GCs) in sheep. We identified the key BMP-6 receptors, activin receptor-like kinase(ALK-6), and bone morphogenetic protein receptor type 2 (BMPRII) in sheep follicles using immunohistochemistry (IHC) and immunofluorescence (IF). Both ALK-6 and BMPRII were expressed in the GC layer, GC membranes, and cytoplasm. We evaluated ALK-6 and BMPRII expression at the follicular development stage using quantitative real-time PCR and western blotting to detect sheep GCs from large, medium, and small follicles (diameters of ≥5, 2-5, and ≤2 mm, respectively). The mRNA abundance and protein expression of ALK-6 and BMPRII were significantly higher in GCs from large follicles compared to those in GCs from small follicles (P < 0.05) and were the lowest in GCs from medium follicles. To assess whether DHT affects ALK-6 and BMPRII expression in sheep GCs, we cultured GCs from large follicles in vitro then incubated them with DHT (10-11, 10-9, 10-7 M). We found that 10-7-M DHT significantly inhibited ALK-6 and BMPRII mRNA and protein (P < 0.05). We further explored whether DHT regulates ALK-6 and BMPRII through the nuclear androgen receptor (AR) pathway and found that 10-6-M flutamide, a non-selective androgen inhibitor, partially relieved the inhibitory effect of 10-7-M DHT on ALK-6 and BMPRII expression. Thus, GCs in sheep antral follicles differentially expressed ALK-6 and BMPRII at various stages, indicating that BMP-6 plays different roles to some extent during the development of antral follicles, and that high concentrations of DHT can inhibit the expression of ALK-6 and BMPRII via the androgen receptor pathway in sheep GCs. The present study aimed to determine the expression of the main BMP-6-related main receptors, namely, ALK-6 and BMPRII, during the development of GCs in sheep antral follicles and a potential mechanism of DHT regulation in sheep GCs. Our findings lay a foundation for the further exploration of the effects of ovarian BMP-6 expression on follicular development.

Epigenetic Silencing of BMP6 by the SIN3A-HDAC1/2 Repressor Complex Drives Melanoma Metastasis via FAM83G/PAWS1.

Aberrant epigenetic transcriptional regulation is linked to metastasis, a primary cause of cancer-related death. Dissecting the epigenetic mechanisms controlling metastatic progression may uncover important insights to tumor biology and potential therapeutic targets. Here, we investigated the role of the SIN3A histone deacetylase 1 and 2 (SIN3A-HDAC1/2) complex in cancer metastasis. Using a mouse model of melanoma metastasis, we found that the SIN3A-HDAC1/2 transcription repressor complex silences BMP6 expression, causing increased metastatic dissemination and tumor growth via suppression of BMP6-activated SMAD5 signaling. We further discovered that FAM83G/PAWS1, a downstream effector of BMP6-SMAD5 signaling, contributes critically to metastatic progression by promoting actin-dependent cytoskeletal dynamics and cell migration. Pharmacologic inhibition of the SIN3A-HDAC1/2 complex reduced the numbers of melanoma cells in the circulation and inhibited metastatic tumor growth by inducing disseminated cell dormancy, highlighting the SIN3A-HDAC1/2 repressor complex as a potential therapeutic target for blocking cancer metastasis. IMPLICATIONS: This study identifies the novel molecular links in the metastatic progression to target cytoskeletal dynamics in melanoma and identifies the SIN3A-HDAC1/2 complex and FAM83G/PAWS1 as potential targets for melanoma adjuvant therapy.

The effect of cellular and physiological indicators on gender determination.

Gender determination, in addition to having special value to parents, has particular importance in sex-linked diseases. This study aimed to investigate the cellular indicators (i.e. BMP-6 protein and PPAR? protein expression levels in granulosa cells) and the physiological indicators on gender determination. For this purpose, on 68 infertile patients referred to the clinic, ovarian stimulation was performed by different protocols and then ruptured by different HCG. Follow-up of patients was performed after they became pregnant after five months. U/S was done for knowing the gender of the baby then after labor rechecked another time. Also, granulosa-luteal cells (GLCs) were isolated from the follicular fluid of 68 women participating in the study. BMP-6 protein and PPAR? protein were measured using Western blotting. Results showed that the total number of delivered babies was 68, 41 males (60.3%) and 27 females (39.7%). About physiological indicators results, there was no significant association between the age of the mother and sex of the baby (P=0.934). No significant association was detected between the month during which the conception occurred and the sex of the baby (P=0.734). The same result was obtained for the follicle side (P=0.236), and follicle size (P=0.659), there was no significant association between the sex of the baby with the following factors: protocol of treatment (P=0.417), IVF after HCG (P=0.237), HCG type (P=0.572), parity (P=0.282), and type of infertility (P=0.376). The cellular indicators results showed that the BMP-6 protein level in granulosa cells of mothers with daughters was almost twice as high as mothers with sons (P=0.043). But there was no significant difference between mothers with daughters and mothers with sons in PPAR? protein level (P=0.12). It can be concluded that except for BMP-6 protein level, none of the cellular and physiological indicators affects gender determination. Therefore, this cell indicator can probably be evaluated as an effective indicator in determining gender.

Bone morphogenetic protein 6-mediated crosstalk between endothelial cells and hepatocytes recapitulates the iron-sensing pathway in vitro.

Liver sinusoidal endothelial cell-derived bone morphogenetic protein 6 (BMP6) and the BMP6-small mothers against decapentaplegic homolog (SMAD) signaling pathway are essential for the expression of hepcidin, the secretion of which is considered the systemic master switch of iron homeostasis. However, there are continued controversies related to the strong and direct suppressive effect of iron on hepatocellular hepcidin in vitro in contrast to in vivo conditions. Here, we directly studied the crosstalk between endothelial cells (ECs) and hepatocytes using in vitro coculture models that mimic hepcidin signaling in vivo. Huh7 cells were directly cocultured with ECs, and EC conditioned media (CM) were also used to culture Huh7 cells and primary mouse hepatocytes. To explore the reactions of ECs to surrounding iron, they were grown in the presence of ferric ammonium citrate and heme, two iron-containing molecules. We found that both direct coculture with ECs and EC-CM significantly increased hepcidin expression in Huh7 cells. The upstream SMAD pathway, including phosphorylated SMAD1/5/8, SMAD1, and inhibitor of DNA binding 1, was induced by EC-CM, promoting hepcidin expression. Efficient blockage of this EC-mediated hepcidin upregulation by an inhibitor of the BMP6 receptor ALK receptor tyrosine kinase 2/3 or BMP6 siRNA identified BMP6 as a major hepcidin regulator in this coculture system, which highly fits the model of hepcidin regulation by iron in vivo. In addition, EC-derived BMP6 and hepcidin were highly sensitive to levels of not only ferric iron but also heme as low as 500 nM. We here establish a hepatocyte-endothelial coculture system to fully recapitulate iron regulation by hepcidin using EC-derived BMP6.

Role of the BMP6 protein in breast cancer and other types of cancer.

The BMP6 protein (Bone Morphogenetic Protein 6) is part of the superfamily of transforming growth factor-beta (TGF-ß) ligands, participates in iron homeostasis, inhibits invasion by increasing adhesions and cell-cell type interactions and induces angiogenesis directly on vascular endothelial cells. BMP6 is coded by a tumor suppressor gene whose subexpression is related to the development and cancer progression; during neoplastic processes, methylation is the main mechanism by which gene silencing occurs. This work presents a review on the role of BMP6 protein in breast cancer (BC) and other types of cancer. The studies carried out to date suggest the participation of the BMP6 protein in the epithelial-mesenchymal transition (EMT) phenotype, cell growth and proliferation; however, these processes are affected in a variable way in the different types of cancer, the methylated CpG sites in BMP6 gene promoter, as well as the interaction with other proteins could be the cause of such variation.