BMP6 Regulates Corneal Epithelial Cell Stratification by Coordinating Their Proliferation and Differentiation and Is Upregulated in Pterygium.

Purpose: Proper balance between cell proliferation and differentiation is essential for corneal epithelial (CE) stratification and homeostasis. Although bone morphogenetic protein-6 (BMP6) is known to be expressed in the CE for over 25 years, its function in this tissue remains unknown. Here, we test the hypothesis that BMP6 promotes CE cell stratification and homeostasis by regulating their proliferation and differentiation. Methods: We employed postnatal day-12 (PN-12), PN-14, PN-20, and PN-90 mouse eyes; human corneal limbal epithelial (HCLE) cells; and ocular surface fibrovascular disease pterygium tissues to evaluate the role of BMP6 in CE proliferation, differentiation, and pathology by RT-qPCR, immunoblots, and/or immunofluorescent staining. Cell proliferation was quantified by immunostaining for Ki67. Results: Coincident with the mouse CE stratification between PN-12 and PN-20, BMP6 was significantly upregulated and the BMP6 antagonist Noggin downregulated. Mature CE retained high BMP6 and low Noggin expression at PN-90. BMP6 and its receptors BMPR1A and BMPR2 were upregulated during in vitro stratification of HCLE cells. Consistent with its anti-proliferative role, exogenous BMP6 suppressed HCLE cell proliferation, downregulated cyclin-D1 and cyclin-D2, and upregulated cell-cycle inhibitors Krüppel-like factor 4 (KLF4) and p21. BMP6 also upregulated the desmosomal cadherins desmoplakin and desmoglein in HCLE cells, consistent with its pro-differentiation role. Human pterygium displayed significant upregulation of BMP6 coupled with downregulation of Noggin and cell-cycle suppressors KLF4 and p21. Conclusions: BMP6 coordinates CE stratification and homeostasis by regulating their proliferation and differentiation. BMP6 is significantly upregulated in human pterygium concurrent with downregulation of Noggin, KLF4, and p21.

Modulatory effects of TGF-ß1 and BMP6 on thecal angiogenesis and steroidogenesis in the bovine ovary.

Angiogenesis plays an integral role in follicular and luteal development and is positively regulated by several intra-ovarian factors including vascular endothelial growth factor A (VEGFA) and fibroblast growth factor 2 (FGF2). Various transforming growth factor-ß (TGF-ß) superfamily members function as intra-ovarian regulators of follicle and luteal function, but their potential roles in modulating ovarian angiogenesis have received little attention. In this study, we used a bovine theca interna culture model (exhibiting characteristics of luteinization) to examine the effects of TGF-ß1 and bone morphogenetic protein 6 (BMP6) on angiogenesis and steroidogenesis. VEGFA/FGF2 treatment promoted endothelial cell network formation but had little or no effect on progesterone and androstenedione secretion or expression of key steroidogenesis-related genes. TGF-ß1 suppressed basal and VEGFA/FGF2-induced endothelial cell network formation and progesterone secretion, effects that were reversed by an activin receptor-like kinase 5 (ALK5) inhibitor (SB-431542). The ALK5 inhibitor alone raised androstenedione secretion and expression of several transcripts including CYP17A1. BMP6 also suppressed endothelial cell network formation under VEGFA/FGF2-stimulated conditions and inhibited progesterone secretion and expression of several steroidogenesis-related genes under basal and VEGFA/FGF2-stimulated conditions. These effects were reversed by an ALK1/2 inhibitor (K02288). Moreover, the ALK1/2 inhibitor alone augmented endothelial network formation, progesterone secretion, androstenedione secretion and expression of several steroidogenesis-related genes. The results indicate dual suppressive actions of both TGF-ß1 and BMP6 on follicular angiogenesis and steroidogenesis. Further experiments are needed to unravel the complex interactions between TGF-ß superfamily signalling and other regulatory factors controlling ovarian angiogenesis and steroidogenesis.

Endothelial Bone Morphogenetic Protein 2 (Bmp2) Knockout Exacerbates Hemochromatosis in Homeostatic Iron Regulator (Hfe) Knockout Mice but not Bmp6 Knockout Mice.

BACKGROUND AND AIMS: Bone morphogenetic proteins BMP2 and BMP6 play key roles in systemic iron homeostasis by regulating production of the iron hormone hepcidin. The homeostatic iron regulator (HFE) also regulates hepcidin through a mechanism that intersects with the BMP-mothers against decapentaplegic homolog 1/5/8 (SMAD1/5/8) pathway. However, the relative roles of BMP2 compared with BMP6 and whether HFE regulates hepcidin through a BMP2-dependent mechanism remain uncertain. APPROACH AND RESULTS: We therefore examined the iron phenotype of mice deficient for both Bmp2 and Bmp6 or both Bmp2 and Hfe compared with single knockout (KO) mice and littermate controls. Eight-week-old double endothelial Bmp6/Bmp2 KO mice exhibited a similar degree of hepcidin deficiency, serum iron overload, and tissue iron overload compared with single KO mice. Notably, dietary iron loading still induced liver SMAD5 phosphorylation and hepcidin in double Bmp6/endothelial Bmp2 KO mice, although no other BMP ligand mRNAs were increased in the livers of double KO mice, and only Bmp6 and Bmp2 mRNA were induced by dietary iron loading in wild-type mice. In contrast, double Hfe/endothelial Bmp2 KO mice exhibited reduced hepcidin and increased extrahepatic iron loading compared to single Hfe or endothelial Bmp2 KO mice. Liver phosphorylated SMAD5 and the SMAD1/5/8 target inhibitor of DNA binding 1 (Id1) mRNA were also reduced in double Hfe/endothelial Bmp2 KO compared with single endothelial Bmp2 KO female mice. Finally, hepcidin and Id1 mRNA induction by homodimeric BMP2, homodimeric BMP6, and heterodimeric BMP2/6 were blunted in Hfe KO primary hepatocytes. CONCLUSIONS: These data suggest that BMP2 and BMP6 work collaboratively to regulate hepcidin expression, that BMP2-independent and BMP6-independent SMAD1/5/8 signaling contributes a nonredundant role to hepcidin regulation by iron, and that HFE regulates hepcidin at least in part through a BMP2-independent but SMAD1/5/8-dependent mechanism.

Nrf2 controls iron homeostasis in haemochromatosis and thalassaemia via Bmp6 and hepcidin.

Iron is critical for life but toxic in excess because of iron-catalysed formation of pro-oxidants that cause tissue damage in a range of disorders. The Nrf2 transcription factor orchestrates cell-intrinsic protective antioxidant responses, and the peptide hormone hepcidin maintains systemic iron homeostasis, but is pathophysiologically decreased in haemochromatosis and beta-thalassaemia. Here, we show that Nrf2 is activated by iron-induced, mitochondria-derived pro-oxidants and drives Bmp6 expression in liver sinusoid endothelial cells, which in turn increases hepcidin synthesis by neighbouring hepatocytes. In Nrf2 knockout mice, the Bmp6-hepcidin response to oral and parenteral iron is impaired and iron accumulation and hepatic damage are increased. Pharmacological activation of Nrf2 stimulates the Bmp6-hepcidin axis, improving iron homeostasis in haemochromatosis and counteracting the inhibition of Bmp6 by erythroferrone in beta-thalassaemia. We propose that Nrf2 links cellular sensing of excess toxic iron to control of systemic iron homeostasis and antioxidant responses, and may be a therapeutic target for iron-associated disorders.

BMP6 increases TGF-ß1 production by up-regulating furin expression in human granulosa-lutein cells.

Bone morphogenetic protein 6 (BMP6) and transforming growth factor-ß1 (TGF-ß1) are key intraovarian regulators that play essential roles in regulating mammalian follicular function and promoting oocyte maturation. Furin, a member of the subtilisin-like proprotein convertase family, promotes the activation of diverse functional proteins by cleaving protein precursors in the secretory pathway. The aim of this study was to investigate the effect and underlying molecular mechanisms by which BMP6 regulates the expression of furin to increase TGF-ß1 production. Primary and immortalized (SVOG) human granulosa-lutein (hGL) cells were used as study models. Our results show that BMP6 significantly up-regulated the expression of furin and increased the production of TGF-ß1 in hGL cells. Using dual inhibition approaches (kinase receptor inhibitors and small interfering RNA-targeted knockdown), we demonstrate that both activin receptor-like (ALK)2 and ALK3 are involved in the BMP6-induced up-regulation of furin. Additionally, knockdown of furin abolished BMP6-induced increases in TGF-ß1 production. Moreover, knockdown of endogenous SMAD4 reversed the BMP6-induced increase in furin expression. These results indicate that the ALK2/3-mediated canonical SMAD signaling pathway is required for the stimulatory effect of BMP6 on furin expression, which in turn increases the production of TGF-ß1 in hGL cells. Our findings provide insights into the molecular interactions and mechanisms of two intrafollicular growth factors in hGL cells.

Iron, erythropoietin, and inflammation regulate hepcidin in Bmp2-deficient mice, but serum iron fails to induce hepcidin in Bmp6-deficient mice.

The bone morphogenetic protein (BMP)-SMAD signaling pathway is a key transcriptional regulator of hepcidin in response to tissue iron stores, serum iron, erythropoietic drive and inflammation to increase the iron supply when needed for erythropoiesis, but to prevent the toxicity of iron excess. Recently, BMP2 was reported to play a non-redundant role in hepcidin regulation in addition to BMP6. Here, we used a newly validated BMP2 ELISA assay and mice with a global or endothelial conditional knockout (CKO) of Bmp2 or Bmp6 to examine how BMP2 is regulated and functionally contributes to hepcidin regulation by its major stimuli. Erythropoietin (EPO) did not influence BMP2 expression in control mice, and still suppressed hepcidin in Bmp2 CKO mice. Lipopolysaccharide (LPS) reduced BMP2 expression in control mice, but still induced hepcidin in Bmp2 CKO mice. Chronic dietary iron loading that increased liver iron induced BMP2 expression, whereas acute oral iron gavage that increased serum iron without influencing liver iron did not impact BMP2. However, hepcidin was still induced by both iron loading methods in Bmp2 CKO mice, although the degree of hepcidin induction was blunted relative to control mice. Conversely, acute oral iron gavage failed to induce hepcidin in Bmp6 -/- or CKO mice. Thus, BMP2 has at least a partially redundant role in hepcidin regulation by serum iron, tissue iron, inflammation and erythropoietic drive. In contrast, BMP6 is absolutely required for hepcidin regulation by serum iron.

A novel role of bone morphogenetic protein 6 (BMP6) in glucose homeostasis.

AIMS: Bone morphogenetic proteins (BMPs) are involved in the development and homeostasis of multiple organs and tissues. There has been a significant focus on understanding the role of BMPs in pancreatic ß-cell dysfunction associated with type 2 diabetes (T2D). Our objective was to investigate the relationship between BMP6 and glucose homeostasis. METHODS: Ob/ob mice were treated with BMP6 for 6 days and analyzed for insulin release, body weight, lipid parameters and glucose tolerance. Quantitative real-time PCR, chromatin immunoprecipitation and glucose output assays were used to assess BMP6 effect on gluconeogenesis in rat hepatoma H4IIE cells. Specificity of BMP6 receptors was characterized by the utilization of various receptor Fc fusion proteins in luciferase reporter gene and glucose output assays in INS1 and H4IIE cells. RESULTS: Treatment of ob/ob mice with BMP6 for 6 days resulted in a reduction of circulating glucose and lipid levels, followed by a significantly elevated plasma insulin level in a dose-dependent manner. In addition, BMP6 improved the glucose excursion during an oral glucose tolerance test, lowering the total glycemic response by 21%. In rat H4IIE hepatoma cells, BMP6 inhibited gluconeogenesis and glucose output via downregulation the PepCK expression. Moreover, BMP6 inhibited glucose production regardless of the presence of cAMP, antagonizing its glycogenolytic effect. BMP6 acted on pancreatic and liver cells utilizing Alk3, Alk6 and ActRIIA serine/threonine kinase receptors. CONCLUSIONS: Collectively, we demonstrate that BMP6 improves glycaemia in T2D mice and regulates glucose metabolism in hepatocytes representing an exciting prospect for future treatments of diabetes.

An intronic enhancer of Bmp6 underlies evolved tooth gain in sticklebacks.

Threespine stickleback fish offer a powerful system to dissect the genetic basis of morphological evolution in nature. Marine sticklebacks have repeatedly invaded and adapted to numerous freshwater environments throughout the Northern hemisphere. In response to new diets in freshwater habitats, changes in craniofacial morphology, including heritable increases in tooth number, have evolved in derived freshwater populations. Using a combination of quantitative genetics and genome resequencing, here we fine-mapped a quantitative trait locus (QTL) regulating evolved tooth gain to a cluster of ten QTL-associated single nucleotide variants, all within intron four of Bone Morphogenetic Protein 6 (Bmp6). Transgenic reporter assays revealed this intronic region contains a tooth enhancer. We induced mutations in Bmp6, revealing required roles for survival, growth, and tooth patterning. Transcriptional profiling of Bmp6 mutant dental tissues identified significant downregulation of a set of genes whose orthologs were previously shown to be expressed in quiescent mouse hair stem cells. Collectively these data support a model where mutations within a Bmp6 intronic tooth enhancer contribute to evolved tooth gain, and suggest that ancient shared genetic circuitry regulates the regeneration of diverse vertebrate epithelial appendages including mammalian hair and fish teeth.

Spatial regulation of bone morphogenetic proteins (BMPs) in postnatal articular and growth plate cartilage.

Articular and growth plate cartilage both arise from condensations of mesenchymal cells, but ultimately develop important histological and functional differences. Each is composed of three layers-the superficial, mid and deep zones of articular cartilage and the resting, proliferative and hypertrophic zones of growth plate cartilage. The bone morphogenetic protein (BMP) system plays an important role in cartilage development. A gradient in expression of BMP-related genes has been observed across growth plate cartilage, likely playing a role in zonal differentiation. To investigate the presence of a similar expression gradient in articular cartilage, we used laser capture microdissection (LCM) to separate murine growth plate and articular cartilage from the proximal tibia into their six constituent zones, and used a solution hybridization assay with color-coded probes (nCounter) to quantify mRNAs for 30 different BMP-related genes in each zone. In situ hybridization and immunohistochemistry were then used to confirm spatial expression patterns. Expression gradients for Bmp2 and 6 were observed across growth plate cartilage with highest expression in hypertrophic zone. However, intracellular BMP signaling, assessed by phospho-Smad1/5/8 immunohistochemical staining, appeared to be higher in the proliferative zone and prehypertrophic area than in hypertrophic zone, possibly due to high expression of Smad7, an inhibitory Smad, in the hypertrophic zone. We also found BMP expression gradients across the articular cartilage with BMP agonists primarily expressed in the superficial zone and BMP functional antagonists primarily expressed in the deep zone. Phospho-Smad1/5/8 immunohistochemical staining showed a similar gradient. In combination with previous evidence that BMPs regulate chondrocyte proliferation and differentiation, the current findings suggest that BMP signaling gradients exist across both growth plate and articular cartilage and that these gradients may contribute to the spatial differentiation of chondrocytes in the postnatal endochondral skeleton.

BMP-6 modulates somatostatin effects on luteinizing hormone production by gonadrotrope cells.

The effects of somatostatin analogs and roles of BMP-6 in the regulation of luteinizing hormone (LH) secretion were investigated using mouse gonadotrope LßT2 cells. LH mRNA expression and LH secretion induced by GnRH were suppressed by treatments with somatostatin analogs, including octreotide and pasireotide, in LßT2 cells. Of note, the inhibitory effects of somatostatin analogs on LH secretion were enhanced by the action of BMP-6. BMP-6 increased the expression levels of somatostatin receptor (SSTR)5, suggesting that BMP-6 upregulates SSTR activity that leads to reduction of GnRH-induced LH secretion. In addition, GnRH-induced phosphorylation of MAPKs including ERK, but not P38 or SAPK, was suppressed by pasireotide in the presence of BMP-6. Given that each inhibitor of ERK, JNK or P38 signaling suppressed GnRH-induced LH transcription, MAPKs are individually involved in the induction of LH production by LßT2 cells. Somatostatin analogs also impaired BMP-6-induced Smad1/5/8 phosphorylation by suppressing BMPRs and augmenting Smad6/7 expression. Collectively, the results indicate that somatostatin analogs have dual effects on the modulation of GnRH-induced MAPK signaling and BMP activity. The pituitary BMP system may play a regulatory role in GnRH-induced LH secretion by tuning the responsiveness to somatostatin analogs in gonadotrope cells.

RE1 silencing transcription factor/neuron-restrictive silencing factor regulates expansion of adult mouse subventricular zone-derived neural stem/progenitor cells in vitro.

Adult neural stem cell (aNSC) activity is tuned by external stimuli through the recruitment of transcription factors. This study examines the RE1 silencing transcription factor (REST) in neural stem/progenitor cells isolated from the subventricular zone of adult mouse brain and provides the first extensive characterization of REST-mediated control of the cellular and molecular properties. This study shows that REST knockdown affects the capacity of progenitor cells to generate neurospheres, reduces cell proliferation, and triggers cell differentiation despite the presence of growth factors. Genome- and transcriptome-wide analyses show that REST binding sites are significantly enriched in genes associated with synaptic transmission and nervous system development and function. Seeking candidate regulators of aNSC function, this study identifies a member of the bone morphogenetic protein (BMP) family, BMP6, the mRNA and protein of which increased after REST knockdown. The results of this study extend previous findings, demonstrating a reciprocal control of REST expression by BMPs. Administration of exogenous BMP6 inhibits aNSC proliferation and induces the expression of the astrocytic marker glial fibrillary acidic protein, highlighting its antimitogenic and prodifferentiative effects. This study suggests that BMP6 produced in a REST-regulated manner together with other signals can contribute to regulation of NSC maintenance and fate.

[Coupling and communication between bone cells].

Bone is constantly renewed by the balanced action of osteoblastic bone formation and osteoclastic bone resorption both of which mainly occur at the bone surface. This restructuring process called "bone remodeling" is important not only for normal bone mass and strength, but also for mineral homeostasis. Coupling has been understood as a balanced induction of osteoblastic bone formation in response to osteoclastic bone resorption. An imbalance of this coupling is often linked to various bone diseases. TGF-ß and IGF released from bone matrix during osteoclastic bone resorption are the favored candidates as classical coupling factor. Recently, several reports suggest that osteoclast-derived molecules/cytokines (clastokine) mediate directional signaling between osteoblasts and osteoclasts into the bone microenvironment. Thus, the elucidation of the regulatory mechanisms involved in bone cell communication and coupling is critical for a deeper understanding of the skeletal system in health and disease.

Role of oocyte-derived paracrine factors in follicular development.

Mammalian oocytes secrete transforming growth factor ß (TGF-ß) superfamily proteins, such as growth differentiation factor 9 (GDF9), bone morphogenetic protein 6 (BMP6) and BMP15, and fibroblast growth factors (FGFs). These oocyte-derived paracrine factors (ODPFs) play essential roles in regulating the differentiation and function of somatic granulosa cells as well as the development of ovarian follicles. In addition to the importance of individual ODPFs, emerging evidence suggests that the interaction of ODPF signals with other intra-follicular signals, such as estrogen, is critical for folliculogenesis. In this review, we will discuss the current understanding of the role of ODPFs in follicular development with an emphasis on their interaction with estrogen signaling in regulation of the differentiation and function of granulosa cells.

The role of bone morphogenetic protein 6 in accumulation and regulation of neutrophils in the human ovary.

Bone morphogenetic protein (BMP) cytokine is known to regulate ovulation, as BMP-6 null mice exhibit a decrease in the number of ovulatory follicles without effect on either the morphology or the dynamics of follicular development. In the present study, the role of BMP-6 in ovulatory process was investigated using human granulosa-lutein cells (GCs). Granulosa-lutein cells, obtained from in vitro fertilization patients, were cultured with BMP-6 followed by RNA extraction. The neutrophil-chemotactic activity of the supernatant of cultured GC was investigated. Bone morphogenetic protein 6 significantly increased growth-regulated oncogene α (GRO-α) messenger RNA (mRNA) and protein expression in GC. In the neutrophil-chemotaxis assay, the GC supernatant cultured with BMP-6 attracted more neutrophils than control samples, which was negated with anti-GRO-α neutralizing antibody. Bone morphogenetic protein 6 also suppressed the relative expression of the protease inhibitors, secretory leukocyte peptidase inhibitor, and whey acid protein 14 mRNA in GC. Bone morphogenetic protein 6 might play a role in ovulation by increasing the accumulation of neutrophils in the ovulatory follicle and suppressing the effect of protease inhibitors.

Mechanism of pro-tumorigenic effect of BMP-6: neovascularization involving tumor-associated macrophages and IL-1a.

INTRODUCTION. Overexpression of bone morphogenetic protein-6 (BMP-6) has been reported in human prostate cancer tissues. Previously we have demonstrated that BMP-6 enhances prostate cancer growth in mice and not in tissue culture. Herein, we have investigated the mechanism of BMP-6's pro-tumorigenic effect in prostate cancer. METHODS. Tramp C2 murine and LNCaP human prostate cancer cell lines were co-cultured with RAW 264.7 and THP-1 cells, respectively. IL-1a knockout mice were used to confirm the role of BMP-6/IL-1a loop in vivo. Lastly, conditional macrophage null mice cd11b-DTR was used. RESULTS. The results demonstrated that BMP-6 induced the expression of IL-1a in macrophages via a cross-talk between NF-kB1 p50 and Smad1. When endothelial cells were treated with conditioned media harvested from macrophages incubated with BMP-6, tube formation was detected. In the presence of IL-1a neutralizing antibody, endothelial tube formation was blocked. In vivo, tumor growth and neovascularization decreased significantly when BMP-6 was expressed in IL-1a knockout and conditional macrophage-null mice. CONCLUSIONS. Prostate cancer-derived BMP-6 stimulates tumor-associated macrophages to produce IL-1a through a crosstalk between Smad1 and NF-kB1; IL-1a, in turn, promotes angiogenesis and prostate cancer growth.

Liver iron modulates hepcidin expression during chronically elevated erythropoiesis in mice.

UNLABELLED: The liver-derived peptide hepcidin controls the balance between iron demand and iron supply. By inhibiting the iron export activity of ferroportin, hepcidin modulates iron absorption and delivery from the body's stores. The regulation of hepcidin, however, is not completely understood and includes a variety of different signals. We studied iron metabolism and hepcidin expression in mice constitutively overexpressing erythropoietin (Epo) (Tg6 mice), which leads to excessive erythropoiesis. We observed a very strong down-regulation of hepcidin in Tg6 mice that was accompanied by a strong increase in duodenal expression of ferroportin and divalent metal tranporter-1, as well as enhanced duodenal iron absorption. Despite these compensatory mechanisms, Tg6 mice displayed marked circulating iron deficiency and low levels of iron in liver, spleen, and muscle. To elucidate the primary signal affecting hepcidin expression during chronically elevated erythropoiesis, we increased iron availability by either providing iron (thus further increasing the hematocrit) or reducing erythropoiesis-dependent iron consumption by means of splenectomy. Both treatments increased liver iron and up-regulated hepcidin expression and the BMP6/SMAD pathway despite continuously high plasma Epo levels and sustained erythropoiesis. This suggests that hepcidin expression is not controlled by erythropoietic signals directly in this setting. Rather, these results indicate that iron consumption for erythropoiesis modulates liver iron content, and ultimately BMP6 and hepcidin. Analysis of the BMP6/SMAD pathway targets showed that inhibitor of DNA binding 1 (ID1) and SMAD7, but not transmembrane serine protease 6 (TMPRSS6), were up-regulated by increased iron availability and thus may be involved in setting the upper limit of hepcidin. CONCLUSION: We provide evidence that under conditions of excessive and effective erythropoiesis, liver iron regulates hepcidin expression through the BMP6/SMAD pathway.

Arsenite suppression of BMP signaling in human keratinocytes.

Arsenic, a human skin carcinogen, suppresses differentiation of cultured keratinocytes. Exploring the mechanism of this suppression revealed that BMP-6 greatly increased levels of mRNA for keratins 1 and 10, two of the earliest differentiation markers expressed, a process prevented by co-treatment with arsenite. BMP also stimulated, and arsenite suppressed, mRNA for FOXN1, an important transcription factor driving early keratinocyte differentiation. Keratin mRNAs increased slowly after BMP-6 addition, suggesting they are indirect transcriptional targets. Inhibition of Notch1 activation blocked BMP induction of keratins 1 and 10, while FOXN1 induction was largely unaffected. Supporting a requirement for Notch1 signaling in keratin induction, BMP increased levels of activated Notch1, which was blocked by arsenite. BMP also greatly decreased active ERK, while co-treatment with arsenite maintained active ERK. Inhibition of ERK signaling mimicked BMP by inducing keratin and FOXN1 mRNAs and by increasing active Notch1, effects blocked by arsenite. Of 6 dual-specificity phosphatases (DUSPs) targeting ERK, two were induced by BMP unless prevented by simultaneous exposure to arsenite and EGF. Knockdown of DUSP2 or DUSP14 using shRNAs greatly reduced FOXN1 and keratins 1 and 10 mRNA levels and their induction by BMP. Knockdown also decreased activated Notch1, keratin 1 and keratin 10 protein levels, both in the presence and absence of BMP. Thus, one of the earliest effects of BMP is induction of DUSPs, which increases FOXN1 transcription factor and activates Notch1, both required for keratin gene expression. Arsenite prevents this cascade by maintaining ERK signaling, at least in part by suppressing DUSP expression.

Bone morphogenetic proteins regulate differentiation of human promyelocytic leukemia cells.

We investigated the role of bone morphogenetic proteins (BMPs) in suppression of all-trans retinoic acid (ATRA)-mediated differentiation of leukemic promyelocytes. In NB4 and HL60 cell lines, BMPs reduced the percentage of differentiated cells, and suppressed PU.1 and C/EBPε gene expression induced by ATRA. BMP and ATRA synergized in the induction of ID genes, causing suppression of differentiation. In primary acute promyelocytic leukemia bone-marrow samples, positive correlation of PML/RARα and negative of RARα with the expression of BMP-4, BMP-6 and ID genes were found. We concluded that BMPs may have oncogenic properties and mediate ATRA resistance by a mechanism that involves ID genes.

Sustained submicromolar H2O2 levels induce hepcidin via signal transducer and activator of transcription 3 (STAT3).

The peptide hormone hepcidin regulates mammalian iron homeostasis by blocking ferroportin-mediated iron export from macrophages and the duodenum. During inflammation, hepcidin is strongly induced by interleukin 6, eventually leading to the anemia of chronic disease. Here we show that hepatoma cells and primary hepatocytes strongly up-regulate hepcidin when exposed to low concentrations of H(2)O(2) (0.3-6 µM), concentrations that are comparable with levels of H(2)O(2) released by inflammatory cells. In contrast, bolus treatment of H(2)O(2) has no effect at low concentrations and even suppresses hepcidin at concentrations of >50 µM. H(2)O(2) treatment synergistically stimulates hepcidin promoter activity in combination with recombinant interleukin-6 or bone morphogenetic protein-6 and in a manner that requires a functional STAT3-responsive element. The H(2)O(2)-mediated hepcidin induction requires STAT3 phosphorylation and is effectively blocked by siRNA-mediated STAT3 silencing, overexpression of SOCS3 (suppressor of cytokine signaling 3), and antioxidants such as N-acetylcysteine. Glycoprotein 130 (gp130) is required for H(2)O(2) responsiveness, and Janus kinase 1 (JAK1) is required for adequate basal signaling, whereas Janus kinase 2 (JAK2) is dispensable upstream of STAT3. Importantly, hepcidin levels are also increased by intracellular H(2)O(2) released from the respiratory chain in the presence of rotenone or antimycin A. Our results suggest a novel mechanism of hepcidin regulation by nanomolar levels of sustained H(2)O(2). Thus, similar to cytokines, H(2)O(2) provides an important regulatory link between inflammation and iron metabolism.