Bone Morphogenic Protein Signaling Pathway Research Articles

A100 HEREDITARY HEMORRHAGIC TELANGIECTASIA: MORE THAN JUST ANOTHER RECTAL BLEED

Abstract Background Hereditary hemorrhagic telangiectasia (HHT), previously known as Osler-Weber-Rendu syndrome, is an autosomal dominant disorder characterized by widespread telangiectasia and visceral arteriovenous (AV) malformations. Diagnosis of HHT is commonly guided by The Curaçao Diagnostic Criteria. Molecular genetics can help confirm the clinical diagnosis. Genes involved include ENG, ACVRL1, SMAD4, and GDF2, which affect the bone morphogenic protein signaling pathway important in maintaining vascular endothelial integrity. Aims This report aims to review the case of a young male from a rural community in Northern Alberta with a possible new diagnosis of HHT, presenting with rectal bleeding in the setting of family history of telangiectasias. Methods Retrospective review of one patient. Results We report the case of a 33-year-old male who presented with a 1-day history of rectal bleeding (bright red turning to black) with hemoglobin at 116g/L. Past medical history was significant for occasional epistaxis since age 8, previous gastrointestinal bleed and laparotomy with pathology showing small intestinal vascular malformation at the age of 15, and small bowel obstruction secondary to adhesions requiring resection at the age of 26. There was no history of iron deficiency anemia. Family history was positive for a mother who had Von Willebrand disease and partial colectomy at the age of 28 due to bowel telangiectasias. His mother had no formal HHT diagnosis, but it was unclear if molecular genetics had been performed. Inpatient colonoscopy showed no culprit lesions but free-floating blood clots throughout the colon (Image). EGD and push enteroscopy were normal. CT enterography was then performed showing AV fistula in the distal ileum and venous malformations in the jejunum. Video capsule endoscopy followed revealed a few non-bleeding telangiectasia and petechiae. The patient was assessed to have possible HHT by The Curaçao Diagnostic Criteria. He was supplemented with IV iron and started on short-acting octreotide. On discharge, he received funding approval for long-acting octreotide and was referred to the Edmonton HHT clinic for follow up and further screening. Conclusions HHT remains underdiagnosed in both children and adults. Maintaining a strong suspicion and prompt diagnosis via the Curaçao Criteria is crucial for appropriate screening of the multisystem complications which stem from the AV malformations. This multidisciplinary approach can aid in reducing morbidity and mortality. Funding Agencies None

PSXIII-B-20 Variants within Genes Tspan18 and NRG3 Associated with Angular Limb Deformity in Rambouillet Rams

Abstract Incidence of angular limb deformities (ALD) have been described in many species of livestock and companion animals. Young, rapidly growing animals are at an increased risk for developing ALD. Although work has been done to identify the casual genetic basis of ALD, bone deformities are likely influenced by the environment, making clear genetic answers challenging to discover. Rambouillet rams from Wyoming, North Dakota and South Dakota annual ram performance tests have been observed to develop limb abnormalities consistent with ALD. Historical incidence of ALD in test rams has ranged from 3 to 18%. A genome-wide association study (GWAS) was conducted with 342 ram-test animals, including 40 ALD-affected rams and 302 unaffected rams collected over three consecutive years. Significant markers were identified within the genes tetraspanin 18 (TSPAN18; p-value = 4.87e-07) and neuregulin 3 (NRG3; p-value = 8.06e-07). In mice, Tspan18 has been proposed as a regulator of endothelial cell Orai1/Ca2+ signaling. Orai1 has also been shown to mediate osteogenic differentiation via the bone morphogenic protein signaling pathway, which suggests the possibility for TSPAN18 involvement in differentiation of chondrocytes and osteocytes. The gene NRG3 is a member of the neuregulin family, which encodes ligands for epidermal growth factor receptors ERBB3 and ERBB4. The epidermal growth factor receptor system has described roles in the regulation of proliferation and differentiation of osteoblast, chondrocyte and osteoclast cells. Identifying genetic risk factors for ALD has the potential to improve animal welfare and animal production in at-risk breeds. This study proposes TSPAN18 and NRG3 as targets for further research towards identifying genetic risk factors for ALD in Rambouillet sheep of the NW United States.

Association analysis of polymorphisms rs12997 in ACVR1 and rs1043784 in BMP6 genes involved in bone morphogenic protein signaling pathway in primary angle-closure and pseudoexfoliation glaucoma patients of Saudi origin

BackgroundGlaucoma is a polygenic neurodegenerative disease and the second most common cause of blindness in Saudi Arabia. To test the hypothesis that genetic variants in the genes involved in the bone morphogenic protein (BMP) signaling pathway may be associated with glaucoma, we investigated the association between 3′ untranslated region variants, rs12997 in ACVR1 and rs1043784 in BMP6, and primary angle-closure glaucoma (PACG) and pseudoexfoliation glaucoma (PXG).MethodsIn a case-control study, TaqMan® real-time PCR-based genotyping was done in 444 subjects consisting of 250 controls, 101 PACG and 95 PXG cases, and tested for genetic association with glaucoma-types and other clinical phenotypes.ResultsRs12997[G] allele in ACVR1 exhibited significant 2-fold increased risk of PACG (p = 0.005) in women but not in men. Similarly, genotype analysis also showed that subjects carrying rs12997[G/G] genotype were at > 2-fold risk of PACG that remained significant after adjustment for age, sex, and Bonferroni correction in the recessive model. Furthermore, this effect was also significant in women only. In PXG, the rs12997[G/G] genotype showed a significant trend towards increased risk of the disease (OR = 2.04, 95% CI = 0.99–4.18, p = 0.049) but did not survive the Bonferroni correction. Regression analysis showed that rs12997[G/G] genotype was a significant predictor of PACG independent of age, sex, and rs1043784 genotypes. Likewise, age and rs12997[G/G] genotype showed significant effect on PXG outcome. The rs12997[A/G] genotype showed significant association with cup/disc ratio as compared to wild-type (p = 0.005) in PXG. Genotype and allele frequencies of rs1043784 in BMP6 did not show any significant association either with PACG or PXG.ConclusionsOur results suggest that the polymorphism rs12997 in the ACVR1 gene involved in the BMP signaling pathway is significantly associated with PACG and PXG in a Saudi cohort. This is the first study to associate this variant/gene with PACG and PXG. However, further studies would be needed to replicate these findings in a large population-based cohort.

BMPing Up Healing Capacity with FKBP12 Ligand

While the wound healing property of the macrolide FK506 is well known, the underlying mechanism has been elusive. In this issue of Cell Chemical Biology, Peiffer etal. (2019) utilize FKBP12 ligand to demonstrate that wound healing effects of FK506 occur via activation of the BMP (bone morphogenic protein) signaling pathway.

Interplay between microRNAs and Wnt, transforming growth factor-β, and bone morphogenic protein signaling pathways promote osteoblastic differentiation of mesenchymal stem cells.

Osteoblasts are terminally differentiated cells with mesenchymal origins, known to possess pivotal roles in sustaining bone microstructure and homeostasis. These cells are implicated in the pathophysiology of various bone disorders, especially osteoporosis. Over the last few decades, strategies to impede bone resorption, principally by bisphosphonates, have been mainstay of treatment of osteoporosis; however, in recent years more attention has been drawn on bone-forming approaches for managing osteoporosis. MicroRNAs (miRNAs) are a broad category of noncoding short sequence RNA fragments that posttranscriptionally regulate the expression of diverse functional and structural genes in a negative manner. An accumulating body of evidence signifies that miRNAs direct mesenchymal stem cells toward osteoblast differentiation and bone formation through bone morphogenic protein, transforming growth factor-β, and Wnt signaling pathways. MiRNAs are regarded as excellent future therapeutic candidates because of their small size and ease of delivery into the cells. Considering their novel therapeutic significance, this review discusses the main miRNAs contributing to the anabolic aspects of bone formation and illustrates their interactions with corresponding signaling pathways involved in osteoblastic differentiation.

Wounding-induced upregulation of the Bone Morphogenic Protein signaling pathway in Drosophila promotes survival against parasitic nematode infection

The common fruit fly, Drosophila melanogaster is an outstanding model to analyze the regulation of conserved signaling pathways. In this study, we examined whether signaling components in the Bone Morphogenic Protein (BMP) branch of the TGF-β signaling pathway are involved in the response to wounding caused by either sterile injury or infection by parasitic nematodes in D. melanogaster adult flies. We found that following sterile injury, the BMP pathway Type I receptor sax and intracellular transcription factor Mad were substantially upregulated. Also, inactivation of Mad or dpp promoted fly survival and increased antimicrobial peptide gene transcript levels upon sterile injury or H. bacteriophora nematode infection, respectively, but not against the bacterial pathogen Photorhabdus luminescens. Our findings indicate the roles of certain BMP signaling components in the regulation of the fly immune response against sterile injury or nematode infection. In conclusion, this study highlights the ability of D. melanogaster to activate the BMP branch of TGF-β signaling in order to modulate the response to injury in the absence or presence of pathogenic infection.

Identification of a rare BMP pathway mutation in a non-syndromic human brain arteriovenous malformation via exome sequencing

Brain arteriovenous malformations (AVMs) are abnormal connections between arteries and veins that can result in hemorrhagic stroke. A genetic basis for AVMs is suspected, and we investigated potential mutations in a 14-year-old girl who developed a recurrent brain AVM. Whole-exome sequencing (WES) of AVM lesion tissue and blood was performed accompanied by in silico modeling, protein expression observation in lesion tissue and zebrafish modeling. A stop-gain mutation (c.C739T:p.R247X) in the gene SMAD family member 9 (SMAD9) was discovered. In the human brain tissue, immunofluorescent staining demonstrated a vascular predominance of SMAD9 at the protein level. Vascular SMAD9 was markedly reduced in AVM peri-nidal blood vessels, which was accompanied by a decrease in phosphorylated SMAD4, a downstream effector protein of the bone morphogenic protein signaling pathway. Zebrafish modeling (Tg kdrl:eGFP) of the morpholino splice site and translation-blocking knockdown of SMAD9 resulted in abnormal cerebral artery-to-vein connections with morphologic similarities to human AVMs. Orthogonal trajectories of evidence established a relationship between the candidate mutation discovered in SMAD9 via WES and the clinical phenotype. Replication in similar rare cases of recurrent AVM, or even more broadly sporadic AVM, may be informative in building a more comprehensive understanding of AVM pathogenesis.

Silicate-based bioceramics regulating osteoblast differentiation through a BMP2 signalling pathway.

Bioactive materials with osteostimulation properties have the potential to promote bone regeneration. We have found that silicate-based biomaterials have the osteostimulation ability for regeneration of large bone defects; however, the corresponding mechanism is unclear. In this study, we set out to elucidate the potential mechanism of silicate-based biomaterials with osteostimulation ability. A model silicate bioceramic, nagelschmidtite (NAGEL, Ca7P2Si2O16), was applied to study their ionic products on the effect of the Bone morphogenic protein (BMP) signaling pathway for osteoblast MC3T3-E1 as NAGEL has been previously shown to have excellent in vitro and in vivo bone-forming activity. BMP signaling, especially BMP2, is involved in bone formation during mammalian development and exhibits versatile regulatory functions in the body. It is found that NAGEL bioceramics significantly enhance the migration and osteoblastic differentiation of MC3T3-E1. mRNA and protein expression of BMP2 is enhanced by NAGEL bioceramics in a dose-dependent manner. Moreover, NAGEL bioceramics activate the Smad-dependent BMP signaling pathway and induce the activation of the BMP downstream cascade (OCN, OPN and Runx2). The accumulation of phosphorylated-Smad1/5 is induced by NAGEL bioceramics in the MC3T3-E1 cell nucleus. It is further found that NAGEL bioceramic-mediated migration, osteoblastic differentiation and the activation of the BMP downstream cascade are significantly downregulated by inhibition of BMP2 activity. Our results suggest that silicate-based NAGEL bioceramics possess excellent in vitro osteostimulation properties and the possible mechanism of silicate-based biomaterials with distinct osteostimulation may be directly related to the activation of the BMP2 signaling pathway of osteoblasts by release of Si-containing bioactive ionic products.

Orai1 mediates osteogenic differentiation via BMP signaling pathway in bone marrow mesenchymal stem cells

Orai1 is a pore-subunit of store-operated Ca2+ release-activated Ca2+ (CRAC) channel that mediates Ca2+ influx in most non-excitable cells via store-operated Ca2+ entry (SOCE) mechanism. We previously demonstrated that Orai1 is involved in mediating osteogenic potential of mesenchymal stem cells (MSCs), but the underlying mechanism of this function remains unknown. Here, we report that Orai1 mediates osteogenic differentiation via bone morphogenic protein (BMP) signaling pathway in bone marrow MSCs (BMSCs). In osteogenic conditions, BMSCs derived from wild-type mice underwent osteoblastic differentiation and induced mineralization as demonstrated by increased alkaline phosphatase activity and alizarin red S staining, respectively. The expression of Runx2, a master regulator of osteoblast differentiation, and osteogenic differentiation markers were markedly increased in wild-type BMSCs under osteogenic conditions. In contrast, osteogenic conditions failed to induce such effects in BMSCs derived from Orai1-deficient (Orai1−/−) mice, indicating that Orai1 is, in part, necessary for osteogenic differentiation of MSCs. We also found that BMP2 successfully induced phosphorylation of Smad1/5/8, the immediate effector molecules of BMP signaling, in wild-type BMSCs, but failed to do so in Orai1−/− BMSCs. Downstream target genes of BMP signaling pathway were consistently increased by osteogenic conditions in wild-type BMSCs, but not in Orai1−/− BMSCs, suggesting a novel molecular link between Orai1 and BMP signaling pathway in the osteogenic differentiation process. Further functional studies demonstrated that activation of BMP signaling rescues osteogenic differentiation capacity of Orai1−/− BMSCs. In conclusion, Orai1 regulates osteogenic differentiation through BMP signaling, and the Orai1-BMP signaling may be a possible therapeutic target for treating bone-related diseases.

Editorial: Morphogens in the Wiring of the Nervous System.

Morphogens are secreted signaling molecules that play instructive roles to regulate tissue patterning and cell identity in a concentration-dependent fashion during early embryonic development. Four principal mammalian morphogens include retinoic acid, as well as members of the Wingless-int (Wnt) and transforming growth factor beta (TGF-β)/bone morphogenic protein (BMP) and sonic hedgehog (Shh) families. Morphogen gradients control a plethora of developmental events related to specification and differentiation, cell and tissue polarity, growth control and regeneration. In the nervous system, neuronal differentiation, the polarized outgrowth of neuronal projections, the pathfinding of axons toward their target cells, and the recognition of pre- and post-synaptic partner cells are fundamental requisites to allow the assembly of functional synapses. During the last decades, a growing body of evidence gathered from invertebrate and vertebrate model organisms has shown that the same morphogens classically known to orchestrate early embryonic development are also involved in the precise wiring of the nervous system. The aim of this research topic is to highlight the fundamental roles that morphogens play during the establishment of synaptic connectivity. Hence, we have brought together 12 original research articles and eight reviews. They are mainly focused on Wnt (Aviles et al.; Bernis et al.; Berwick and Harvey; Dickins and Salinas; Pinto et al.; Rosso and Inestrosa; Silva-Alvarez et al.; Varela-Nallar and Inestrosa; Aviles et al.; Varela-Nallar et al.), BMP (Gamez et al.; Pinto et al.; Osses and Henriquez), and Shh (Aviles et al.; Reinchisi et al.) signaling pathways. Research also covers the function of signaling cascades activated by other types of morphogens, including the fibroblast growth factors (FGF; Lee and Umemori; Paradiso et al.), the hepatocyte growth factor (HGF) (Bhardwaj et al.), and netrin (Yamagishi et al.). In addition, researchers have contributed with the emerging roles of new molecules, such as the thyroid hormone (Dezonne et al.), SCO-spondin (Vera et al.), and vitamin C (Pastor et al.). Articles are focused on a wide variety of cellular processes involved in the establishment of neuronal connectivity, such as neurogenesis, neuronal specification, and maturation (Berwick and Harvey; Dezonne et al.; Gamez et al.; Pastor et al.; Reinchisi et al.; Rosso and Inestrosa; Varela-Nallar and Inestrosa; Vera et al.; Varela-Nallar et al.; Yamagishi et al.), axonal outgrowth, polarization, and guidance (Aviles et al.; Bernis et al.; Bhardwaj et al.; Pinto et al.; Aviles et al.; Osses and Henriquez), and synapse formation (Dickins and Salinas; Rosso and Inestrosa; Aviles et al.; Osses and Henriquez), either in physiological contexts or in models of diseases affecting the normal function of the nervous system, including epilepsy (Lee and Umemori; Paradiso et al.), Alzheimer's disease (Silva-Alvarez et al.; Varela-Nallar et al.), and amyotrophic lateral sclerosis (Pinto et al.). We are confident that this integrative research topic emphasizes the central and pleiotropic roles played by morphogens during neural development. We therefore hope that the original articles and reviews presented here will inspire future directions of research focusing on the diversity of cell signaling mechanisms controlling the assembly, maintenance and regeneration of the nervous system.

BMP and TGFbeta pathways in human central chondrosarcoma: enhanced endoglin and Smad 1 signaling in high grade tumors

BackgroundAs major regulators of normal chondrogenesis, the bone morphogenic protein (BMP) and transforming growth factor β (TGFB) signaling pathways may be involved in the development and progression of central chondrosarcoma. In order to uncover their possible implication, the aim of this study was to perform a systematic quantitative study of the expression of BMPs, TGFBs and their receptors and to assess activity of the corresponding pathways in central chondrosarcoma.MethodsGene expression analysis was performed by quantitative RT-PCR in 26 central chondrosarcoma and 6 healthy articular cartilage samples. Expression of endoglin and nuclear localization of phosphorylated Smad1/5/8 and Smad2 was assessed by immunohistochemical analysis.ResultsThe expression of TGFB3 and of the activin receptor-like kinase ALK2 was found to be significantly higher in grade III compared to grade I chondrosarcoma. Nuclear phosphorylated Smad1/5/8 and Smad2 were found in all tumors analyzed and the activity of both signaling pathways was confirmed by functional reporter assays in 2 chondrosarcoma cell lines. Immunohistochemical analysis furthermore revealed that phosphorylated Smad1/5/8 and endoglin expression were significantly higher in high-grade compared to low-grade chondrosarcoma and correlated to each other.ConclusionsThe BMP and TGFβ signaling pathways were found to be active in central chondrosarcoma cells. The correlation of Smad1/5/8 activity to endoglin expression suggests that, as described in other cell types, endoglin could enhance Smad1/5/8 signaling in high-grade chondrosarcoma cells. Endoglin expression coupled to Smad1/5/8 activation could thus represent a functionally important signaling axis for the progression of chondrosarcoma and a regulator of the undifferentiated phenotype of high-grade tumor cells.

HLA-B-associated transcript 3 (Bat3/Scythe) negatively regulates Smad phosphorylation in BMP signaling

Members of the transforming growth factor-β (TGF-β) superfamily participate in numerous biological phenomena in multiple tissues, including in cell proliferation, differentiation, and migration. TGF-β superfamily proteins therefore have prominent roles in wound healing, fibrosis, bone formation, and carcinogenesis. However, the molecular mechanisms regulating these signaling pathways are not fully understood. Here, we describe the regulation of bone morphogenic protein (BMP) signaling by Bat3 (also known as Scythe or BAG6). Bat3 overexpression in murine cell lines suppresses the activity of the Id1 promoter normally induced by BMP signaling. Conversely, Bat3 inactivation enhances the induction of direct BMP target genes, such as Id1, Smad6, and Smad7. Consequently, Bat3 deficiency accelerates the differentiation of primary osteoblasts into bone, with a concomitant increase in the bone differentiation markers Runx2, Osterix, and alkaline phosphatase. Using biochemical and cell biological analyses, we show that Bat3 inactivation sustains the C-terminal phosphorylation and nuclear localization of Smad1, 5, and 8 (Smad1/5/8), thereby enhancing biological responses to BMP treatment. At the mechanistic level, we show that Bat3 interacts with the nuclear phosphatase small C-terminal domain phosphatase (SCP) 2, which terminates BMP signaling by dephosphorylating Smad1/5/8. Notably, Bat3 enhances SCP2–Smad1 interaction only when the BMP signaling pathway is activated. Our results demonstrate that Bat3 is an important regulator of BMP signaling that functions by modulating SCP2–Smad interaction.

A role for NRAGE in NF-κB activation through the non-canonical BMP pathway

BackgroundPrevious studies have linked neurotrophin receptor-interacting MAGE protein to the bone morphogenic protein signaling pathway and its effect on p38 mediated apoptosis of neural progenitor cells via the XIAP-Tak1-Tab1 complex. Its effect on NF-κB has yet to be explored.ResultsHerein we report that NRAGE, via the same XIAP-Tak1-Tab1 complex, is required for the phosphorylation of IKK -α/β and subsequent transcriptional activation of the p65 subunit of NF-κB. Ablation of endogenous NRAGE by siRNA inhibited NF-κB pathway activation, while ablation of Tak1 and Tab1 by morpholino inhibited overexpression of NRAGE from activating NF-κB. Finally, cytokine profiling of an NRAGE over-expressing stable line revealed the expression of macrophage migration inhibitory factor.ConclusionModulation of NRAGE expression revealed novel roles in regulating NF-κB activity in the non-canonical bone morphogenic protein signaling pathway. The expression of macrophage migration inhibitory factor by bone morphogenic protein -4 reveals novel crosstalk between an immune cytokine and a developmental pathway.

Knockdown of Matriptase-2 Increases Hepcidin Expression in a BMP-Dependent Manner in Zebrafish Embryos.

Abstract 4050Poster Board III-985Hepatocytes produce hepcidin, a transcriptionally regulated peptide hormone, in response to iron overload, resulting in decreased intestinal iron absorption. Hepcidin levels are inappropriately low in patients with thalassemia major and hemochromatosis. We have been developing the zebrafish embryo as a model to identify hepcidin regulators, which may be exploited to prevent and treat iron overload syndromes. Recently, it has been shown in mammalian models that the membrane-bound serine protease, matriptase-2 (also known as TMPRSS-6), inhibits hepcidin expression by cleaving membrane-bound hemojuvelin (hjv), a bone morphogenic protein (BMP) co-receptor, which is mutated in patients with juvenile hemochromatosis. Previously, we demonstrated that hepcidin expression increases in response to iron loading or activation of the BMP signaling pathway, but that hjv is not required for hepcidin expression in zebrafish embryos. We hypothesized that hepcidin expression in zebrafish embryos is normally hjv-independent, either because of low levels of hjv expression in the liver or because of the presence of matriptase-2. We used whole mount in situ hybridization, quantitative realtime RT-PCR, morpholino knockdowns and overexpression experiments to address these questions. As hjv was undetectable in the zebrafish embryonic liver by in situ hybridization, we used flow cytometry to sort zebrafish embryonic hepatocytes and assess hjv expression by RT-PCR. We found that hjv was weakly expressed in the embryonic liver, but strongly expressed in zebrafish adult liver. We overexpressed zebrafish hjv in human hepatocytes and found that it potentiated the effects of BMP6 to activate a human hepcidin promoter and a BMP response element. In contrast, overexpression of zebrafish hjv in zebrafish embryos failed to increase hepcidin expression. Knockdown of matriptase-2 in zebrafish embryos caused developmental delay and a smaller liver, but increased hepcidin expression, relative to liver size. Furthermore, treatment with the BMP inhibitor dorsomorphin abrogated the increased hepcidin expression associated with matriptase-2 knockdown. Experiments are underway to determine whether zebrafish hjv has little effect on hepcidin expression during embryonic development because of the activity of matriptase-2. Disclosures:No relevant conflicts of interest to declare.

Presynaptic Contributions of Chordin to Hippocampal Plasticity and Spatial Learning

Recently, several evolutionary conserved signaling pathways that play prominent roles in regulating early neurodevelopment have been found to regulate synaptic remodeling in the adult. To test whether adult neuronal expression of bone morphogenic protein (BMP) signaling components also plays a postnatal role in regulating neuronal plasticity, we modulated BMP signaling in mice both in vivo and in vitro by genetic removal of the BMP inhibitor chordin or by perfusing recombinant BMP signaling pathway components onto acute hippocampal slices. Chordin null mice exhibited a significant increase in presynaptic transmitter release from hippocampal neurons, resulting in enhanced paired-pulse facilitation and long-term potentiation. These mice also showed a decreased acquisition time in a water maze test along with less exploratory activity during Y-maze and open-field tests. Perfusion of BMP ligands onto hippocampal slices replicated the presynaptic phenotype of chordin null slices, but bath application of Noggin, another antagonist of BMP signaling pathway, significantly decrease the frequency of miniature EPSCs. These results demonstrate that the BMP signaling pathway contributes to synaptic plasticity and learning likely through a presynaptic mechanism.

Bone Morphogenic Protein (Smad)-Mediated Repression of Proopiomelanocortin Transcription by Interference with Pitx/Tpit Activity

The signaling molecules bone morphogenic protein (BMP) 4 and 2 have been implicated in early organogenesis and cell differentiation of the pituitary. However, the use of different experimental paradigms has led to conflicting interpretations with regard to the action of these factors on differentiation of corticotroph cells and on expression of the proopiomelanocortin (POMC) gene. We have now directly assessed the action of BMP signaling on POMC expression and found that BMP4 represses POMC mRNA levels and promoter activity. This repression appears to be dependent on the classical BMP signaling pathway that involves the activin-like kinase 3/6 receptors and the Smad1/4 transcription factors. The repression is reversed by overexpression of the inhibitory Smads, Smad6 or Smad7. Collectively, the evidence suggests that autocrine BMP signaling may be acting upon AtT-20 cells to set the level of POMC expression. Upon BMP4 stimulation, activated phospho-Smad1 is recruited to the POMC promoter, where it apparently acts through interactions with the Pitx and Tpit transcription factors. It is postulated that these interactions interfere with the transcriptional activity of Pitx and/or Tpit, thus resulting in transcriptional repression.