We investigated the expressions of bone morphogenetic protein-4 (BMP4) and its receptors, bone morphogenetic protein receptor IA (BMPRIA), bone morphogenetic protein receptor IB (BMPRIB) and bone morphogenetic protein receptor II (BMPRII) in the adult rat eye. Interesting differences in expression profile were observed between BMPRIA and BMPRIB in the retina. BMPRIA-like immunoreactivity (IR) was very intensely seen in the photoreceptor layer, while BMPRIB-IR was mainly observed in the other layers. In the cornea, BMP4, BMPRIA, BMPRIB and BMPRII-IRs were abundantly seen in the cell body of basal cells in the corneal epithelium, and endothelium. In the lens, BMP4, BMPRIA, BMPRIB and BMPRII-IRs were observed in epithelial cells, lens cortical fiber cells, however they were not seen in the capsule and the central region of the lens. In the iris and ciliary body, strong BMP4 and BMPRIB-IRs were observed in nonpigmented epithelium. These results suggest that different kinds of BMP signaling should be needed in different areas in the adult eye to keep the shapes, differentiation levels, and functions of various cells.

Cadmium (Cd) is a well-characterized bone toxic agent and can induce bone damage via inhibiting osteogenic differentiation. Bone morphogenetic protein (BMP)/SMAD signaling pathway can mediate osteogenic differentiation, but the association between Cd and BMP/SMAD signaling pathway is yet to be illuminated. To understand what elements of BMPs and SMADs are affected by Cd to influence osteogenic differentiation and if BMPs can be the biomarkers of which Cd-induced osteoporosis, human bone marrow mesenchymal stem cells (hBMSCs) were treated with cadmium chloride (CdCl2) in vitro to detect the expression of BMPs and SMADs, and 134 subjects were enrolled to explore if the BMPs can be potential biomarkers of Cd-associated bone damage. Our results showed that Cd exposure significantly promoted the adipogenic differentiation of hBMSCs and inhibited its osteogenic differentiation by inhibiting the expression of BMP-2/4, SMAD4, and p-SMAD1/5/9 complex. And mediation analyses yielded that BMP-4 mediated 39.32% (95% confidence interval 7.47, 85.00) of the total association between the Cd and the risk of Cd-associated bone damage. Moreover, during differentiation, BMP-4 had the potential to enhance mineralization compared with CdCl2 only group. These results reveal that BMP-4 can be a diagnostic biomarker and therapeutic target for Cd-associated bone damage.

Bone morphogenetic proteins (BMPs) - expressed in the developing retina - are known to be involved in the regulation of cell proliferation and apoptosis in several tumor entities. The objective of this study was to determine the role of the BMP4 pathway in retinoblastoma cells, which are absent in a functional retinoblastoma (RB1) gene. BMP receptors were detected in all retinoblastoma cell lines investigated. A correct transmission of BMP signaling via the Smad1/5/8 pathway could be demonstrated in WERI-Rb1 retinoblastoma cells and application of recombinant human BMP4 resulted in an increase in apoptosis, which to a large extend is caspase independent. Cell proliferation was not affected by BMP4 signaling, although the pRb-related proteins p107 and p130, contributing to the regulation of the same genes, are still expressed. WERI-Rb1 cells exhibit elevated endogenous levels of p21(CIP1) and p53, but we did not detect any increase in p53, p21(CIP1)or p27(KIP1) expression levels. Id proteins became, however, strongly up-regulated upon exogenous BMP4 treatment. Thus, RB1 loss in WERI-Rb1 cells is obviously not compensated for by pRb-independent (e.g. p53-dependent) cell cycle control mechanisms, preventing an anti-proliferative response to BMP4, which normally induces cell cycle arrest.

Bone morphogenetic protein-4 (BMP4) is a member of the transforming growth factor-beta (TGF-beta) superfamily and plays important roles in multiple biological events. Although BMP4 expression has been well described in the early development of the central nervous system (CNS), little information is available on its expression in the adult CNS. Therefore, we investigated BMP4 expression in the adult rat CNS by using immunohistochemistry. BMP4 is intensely expressed in most neurons and their dendrites. In addition, intense BMP4 expression was also observed in the neuropil of the gray matters where high plasticity is reported, such as the molecular layer of the cerebellum and the superficial layer of the superior colliculus. Furthermore, we found that astrocytes also express BMP4 protein. These data indicate that BMP4 is more widely expressed throughout the adult CNS than previously reported, and its continued abundant expression in the adult brain strongly supports the idea that BMP4 plays pivotal roles also in the adult brain.

Bone defects and nonunions are major clinical skeletal problems. Growth factors are commonly used to promote bone regeneration; however, the clinical impact is limited because the factors do not last long at a given site. The introduction of tissue engineering aimed to deter the diffusion of these factors is a promising therapeutic strategy. The purpose of the present study was to evaluate the in vivo osteogenic capability of an engineered bone morphogenetic protein-4 (BMP4) fusion protein.

Nonalcoholic steatohepatitis (NASH) is a state of simple steatosis that progresses to inflammation and liver injury accompanied by ferroptosis. Bone morphogenetic protein 4 (BMP4) plays an important role in adipogenesis and differentiation, as well as in hepatic steatosis and iron regulation. However, the direct impact of BMP4 on NASH remains unclear. In this study, our aim was to investigate the effect of BMP4 on NASH and its underlying mechanism. We first explored BMP4 expression in vivo in mice and patients and in vitro in HepG2 and LO2 cell lines, and then, determined whether ferroptosis occurs in NASH. Further overexpression or inhibition of BMP4 was induced to observe the effect of BMP4 on liver ferroptosis in NASH. BMP4 expression was upregulated in patients and mice with nonalcoholic fatty liver disease, and free fatty acid (FFA)-induced HepG2 and LO2 cell lines. We observed ferroptosis in high-fat diet and high-fructose diet-fed mice and FFA-induced HepG2 and LO2 cell lines. BMP4 overexpressing plasmid was constructed and the HepG2 and LO2 cells were transfected with lentivirus (oe-BMP4), or treated with exogenously added recombinant human BMP4 or BMP antagonist noggin. BMP4 suppressed the markers of hepatic steatosis, liver inflammation, and liver injury. Upregulated BMP4 expression in HepG2 and LO2 cells reduced reactive oxygen species and malondialdehyde content and relieved ferroptosis. Mechanistically, BMP4 overexpression in hepatocytes upregulated the mRNA and protein levels of glutathione peroxidase 4 (GPX4), a central regulator of ferroptosis, while exogenous inhibition of BMP4 by noggin decreased their levels. Immunoprecipitation assays demonstrated a physical interaction between BMP4 and GPX4 in HepG2 and LO2 cells, and confocal imaging confirmed colocalization of BMP4 and GPX4. Consistently, BMP4 overexpression plays an important role in NASH by increasing GPX4 expression, therefore decreasing hepatic ferroptosis. This study proposes BMP4 as a therapeutic target for preventing steatohepatitis.

Corneal injury may cause neovascularization and lymphangiogenesis in cornea which have a detrimental effect to vision and even lead to blindness. Bone morphogenetic protein 4 (BMP4) regulates a variety of biological processes, which is closely relevant to the regulation of corneal epithelium and angiogenesis. Herein, we aimed to evaluate the effect of BMP4 on corneal neovascularization (CNV), corneal lymphangiogenesis (CL), corneal epithelial repair, and the role of BMP4/Smad pathway in these processes.

Idiopathic pulmonary fibrosis (IPF), ie, usual interstitial pneumonia in histopathology, is a disease characterized by tissue destruction and active areas of fibroproliferation in the lung. Gremlin (Drm), a member of the cysteine knot family of bone morphogenetic protein (BMP) inhibitors, functions to antagonize BMP-4-mediated signals during lung development. We describe here consistent overexpression of gremlin in the lung interstitium of IPF patients. Quantitative real-time reverse transcriptase-polymerase chain reaction analyses revealed considerably higher levels of gremlin mRNA in lung biopsies from IPF patients, the highest level being 35-fold higher compared to controls. Lung fibroblasts isolated from IPF patients also expressed elevated levels of gremlin, which was associated with impaired responsiveness to endogenous and exogenous BMP-4. Transforming growth factor-beta-induced epithelial-to-mesenchymal transition of A549 lung epithelial cells in culture was also associated with induction of gremlin mRNA expression. In addition, A549 cells transfected to overexpress gremlin were more susceptible to transforming growth factor-beta-induced epithelial-to-mesenchymal transition. Gremlin-mediated inhibition of BMP-4 signaling pathways is likely to enhance the fibrotic response and reduce epithelial regeneration in the lung. The overexpression of this developmental gene in IPF may be a key event in the persistence of myofibroblasts in the lung interstitium and provides a potential target for therapeutic intervention.

Bone morphogenetic protein-4 (BMP4) mediates pathological cardiac hypertrophy. Insulin is well-known to promote cardiomyocyte survival in heart diseases. The aim of the present study is to evaluate the effects of insulin on BMP4-induced cardiomyocyte apoptosis. Cell viability and apoptosis were measured by using MTT, live and dead staining, caspase-3 activity assays, and the protein expressions were measured by using western blot technique. Insulin did not elicit cardiomyocyte apoptosis, but antagonized BMP4-induced cardiomyocyte apoptosis. Insulin treatment rapidly activated Akt which was inhibited by Akt inhibitor in cardiomyocytes. Furthermore, Akt inhibitor canceled the anti-apoptotic effects of insulin against BMP4 in cardiomyocytes. In conclusion, insulin prevents BMP4-induced cardiomyocyte apoptosis and the underlying mechanisms include activation of Akt. The present work provides a novel mechanism of the protective effects of insulin in cardiovascular system.

Bone morphogenetic protein-2/4 (BMP2/4) has been recognized as promoters of astrocyte activity. Substantial evidence suggests that BMP2/4 may be elevated and plays a critical role in astrocyte activation upon spinal cord injury. Although neuropathic pain is similarly associated with astrocyte activation, the participation of BMP2/4 in this regard still remains unclear.

Proliferative vitreoretinopathy (PVR), a serious vision-threatening complication of retinal detachment (RD), is characterized by the formation of contractile fibrotic membranes, in which epithelial-mesenchymal transition (EMT) of the retinal pigment epithelium (RPE) is a major event. Recent studies suggest an important role of bone morphogenetic protein 4 (BMP4) in the suppression of fibrosis. In this study, we aimed to investigate the role of BMP4 in the pathological process of PVR, particularly in the EMT of RPE cells. We found that BMP4 and its receptors were co-labelled with cytokeratin and α-SMA positive cells within the PVR membrane. Moreover, the mRNA and protein expression levels of BMP4 were decreased whereas BMP4 receptors ALK2, ALK3 and ALK6 were increased during TGF-β-induced EMT in primary RPE cells. Exogenous BMP4 inhibited TGF-β-induced epithelial marker down-regulation, as well as mesenchymal marker up-regulation at both the mRNA and protein levels in RPE cells. In addition, BMP4 treatment attenuated the TGF-β-induced gel contraction, cell migration and Smad2/3 phosphorylation. However, knockdown of endogenous BMP4 stimulated changes in EMT markers. Our results confirm the hypothesis that BMP4 might inhibit TGF-β-mediated EMT in RPE cells via the Smad2/3 pathway and suppress contraction. This might represent a potential treatment for PVR.

Permanent hearing loss is often a result of damage to cochlear hair cells, which mammals are unable to regenerate. Non-mammalian vertebrates such as birds replace damaged hair cells and restore hearing function, but mechanisms controlling regeneration are not understood. The secreted protein bone morphogenetic protein 4 (BMP4) regulates inner ear morphogenesis and hair cell development. To investigate mechanisms controlling hair cell regeneration in birds, we examined expression and function of BMP4 in the auditory epithelia (basilar papillae) of chickens of either sex after hair cell destruction by ototoxic antibiotics. In mature basilar papillae, BMP4 mRNA is highly expressed in hair cells, but not in hair cell progenitors (supporting cells). Supporting cells transcribe genes encoding receptors for BMP4 (BMPR1A, BMPR1B, and BMPR2) and effectors of BMP4 signaling (ID transcription factors). Following hair cell destruction, BMP4 transcripts are lost from the sensory epithelium. Using organotypic cultures, we demonstrate that treatments with BMP4 during hair cell destruction prevent supporting cells from upregulating expression of the pro-hair cell transcription factor ATOH1, entering the cell cycle, and fully transdifferentiating into hair cells, but they do not induce cell death. By contrast, noggin, a BMP4 inhibitor, increases numbers of regenerated hair cells. These findings demonstrate that BMP4 antagonizes hair cell regeneration in the chicken basilar papilla, at least in part by preventing accumulation of ATOH1 in hair cell precursors.

Subcortical small vessel disease (SVD) is characterized by white matter damage resulting from arteriolosclerosis and chronic hypoperfusion. Transforming growth factor beta 1 (TGFB1) is dysregulated in the hereditary SVD, CARASIL (cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy). However, very little is known about the role of the largest group in the TGFB superfamily - the bone morphogenetic proteins (BMPs) - in SVD pathogenesis. The aim of this study was to characterize signaling abnormalities of BMPs in sporadic SVD. We examined immunostaining of TGFB1 and BMPs (BMP2/BMP4/BMP6/BMP7/BMP9) in a total of 19 post-mortem human brain samples as follows: 7 SVD patients (4 males, 76-90 years old); 6 Alzheimer's disease (AD) patients (2 males, 67-93 years old) and 6 age-matched disease controls (3 males, 68-78 years old). We subsequently investigated the effects of oxygen-glucose deprivation and BMP4 addition on cultured cells. Furthermore, adult mice were subjected to chronic cerebral hypoperfusion using bilateral common carotid artery stenosis, followed by continuous intracerebroventricular infusion of the BMP antagonist, noggin. In the SVD cases, BMP4 was highly expressed in white matter pericytes. Oxygen-glucose deprivation induced BMP4 expression in cultured pericytes in vitro. Recombinant BMP4 increased the number of cultured endothelial cells and pericytes and converted oligodendrocyte precursor cells into astrocytes. Chronic cerebral hypoperfusion in vivo also upregulated BMP4 with concomitant white matter astrogliogenesis and reduced oligodendrocyte lineage cells, both of which were suppressed by intracerebroventricular noggin infusion. Our findings suggest ischemic white matter damage evolves in parallel with BMP4 upregulation in pericytes. BMP4 promotes angiogenesis, but induces astrogliogenesis at the expense of oligodendrocyte precursor cell proliferation and maturation, thereby aggravating white matter damage. This may explain white matter vulnerability to chronic hypoperfusion. The regulation of BMP4 signaling is a potential therapeutic strategy for treating SVD.

Bone morphogenetic proteins (BMPs) comprise one of the largest subgroups in the TGF-β ligand superfamily. We have identified a functional BMP system equipped with the ligand (BMP4), receptors (BMP type II receptor, BMP type IA receptor, also called ALK3) and the signaling proteins, namely the mothers against decapentaplegic homologs 1, 4, and 5 in the human adrenal gland and the human adrenocortical cell line H295R. Microarray, quantitative RT-PCR, and immunohistochemistry confirmed that BMP4 expression was highest in the adrenal zona glomerulosa followed by the zona fasciculata and zona reticularis. Treatment of H295R cells with BMP4 caused phosphorylation of the mothers against decapentaplegic and a profound decrease in synthesis of the C19 steroids dehydroepiandrosterone (DHEA), DHEA sulfate, and androstenedione. Administration of BMP4 to cultures of H295R cells also caused a profound decrease in the mRNA and protein levels of 17α-hydroxylase/17,20-lyase (CYP17A1 and P450c17, respectively) but no significant effect on the mRNA levels of cholesterol side-chain cleavage cytochrome P450 (CYP11A1) or type 2 3β-hydroxysteroid dehydrogenase (HSD3B2). Furthermore, Noggin (a BMP inhibitor) was able to reverse the negative effects of BMP4 with respect to both CYP17A1 transcription and DHEA secretion in the H295R cell line. Collectively the present data suggest that BMP4 is an autocrine/paracrine negative regulator of C19 steroid synthesis in the human adrenal and works by suppressing P450c17.

This Classic Article is a reprint of the original work by Marshall R. Urist and Basil S. Strates, Bone Morphogenetic Protein. An accompanying biographical sketch of Marshall R. Urist, MD is available at DOI 10.1007/s11999-009-1067-4; a second Classic Article is available at DOI 10.1007/s11999-009-1069-2; and a third Classic Article is available at DOI 10.1007/s11999-009-1070-9. The Classic Article is copyright 1971 by Sage Publications Inc. Journals and is reprinted with permission from Urist MR, Strates BS. Bone morphogenetic protein. J Dent Res. 1971;50:1392-1406.

Bone morphogenetic proteins (BMPs) are multifunctional cytokines that elicit pleiotropic effects on biological processes such as cell proliferation, cell differentiation and tissue morphogenesis. With respect to cell proliferation, BMPs can exert either mitogenic or anti-mitogenic activities, depending on the target cells and their context. Here, we report that in low-density cultures of immortalized mammary epithelial cells, BMP-4 did not stimulate cell proliferation by itself. However, when added in combination with suboptimal concentrations of fibroblast growth factor (FGF)-2, FGF-7, FGF-10, epidermal growth factor (EGF) or hepatocyte growth factor (HGF), BMP-4 potently enhanced growth factor-induced cell proliferation. These results reveal a hitherto unsuspected interplay between BMP-4 and growth factors in the regulation of mammary epithelial cell proliferation. We suggest that the ability of BMP-4 to potentiate the mitogenic activity of multiple growth factors may contribute to mammary gland ductal morphogenesis as well as to breast cancer progression.

Biologically active human bone morphogenetic protein-4 (hBMP-4) was successfully produced in a prokaryotic host. For this aim, hBMP-4 cDNA was cloned in Escherichia coli (E. coli) and the protein was produced in a non-active aggregated form. After washing and solubilization, in vitro refolding of the rhBMP-4 monomer was performed using rapid dilution. In this study, different refolding conditions were tested for the dimerization of rhBMP-4 by one-factor-at-a-time variation. The dimerization process was found to be sensitive to pH, protein concentration and the presence of aggregation suppressors. In contrast, redox conditions and ionic strength did not impact refolding as expected. The dimer was separated from the remaining monomer, aggregates and host cell contaminants in a single step using cation-exchange membrane chromatography. The rhBMP-4 dimer produced in E. coli was biologically active as demonstrated by its capability to induce trophoblast differentiation and primitive streak induction of human pluripotent stem cells (hPSCs).

The purpose of this study is to investigate the role of platelet bone morphogenetic proteins (BMP)-4 during vascular inflammation and remodeling in a mouse model of carotid wire injury. Transgenic mice with a platelet-specific deletion of BMP-4 (BMP4Plt-/-) were generated. Intravital microscopy was performed to evaluate leukocyte adhesion to the vessel wall. Expression of adhesion molecules and chemokines were analyzed. Platelet-leukocyte aggregates (PLAs) were evaluated using flow cytometry. For carotid wire injury, BMP4Plt-/- mice were further crossed with LDLr-/- mice (BMP4Plt-/-/LDLr-/-) and fed with a high cholesterol diet for 2-weeks. Carotid wire injury was performed, and re-endothelialization and neointimal formation were evaluated. In comparison to the control mice, stimulation with TNFα resulted in fewer rolling and adherent leukocytes to the vessel wall in the BMP4Plt-/- mice. mRNA and protein expression of P-selectin and adhesion molecules were reduced in the aorta of the BMP4Plt-/- mice. In platelets from the BMP4Plt-/- mice, the expression of P-selectin was reduced, and fewer PLA formations were measured than in the control mice. Loss of platelet BMP-4 further prevented neointima formation after carotid wire injury. Endothelial regeneration after injury was decelerated in the BMP4Plt-/- mice, and confirmed in-vitro, where the deletion of platelet BMP-4 inhibited endothelial cell proliferation and migration. We demonstrate for the first time that platelet BMP-4 is involved during vascular inflammation and remodeling. This is partially mediated by the inhibition of platelet activation, reduced expression of adhesion molecules and inflammatory responses. Our findings identify platelet BMP-4 as a mediator of vascular inflammation in early atherosclerosis and restenosis.

We performed serial analysis of gene expression (SAGE) profiling in mouse chondrogenic ATDC5 cells before and 6 h after the onset of chondrogenesis induced by BMP4. A total of 43,656 SAGE tags (21,875 and 21,781 tags from the uninduced and induced libraries, respectively) were analyzed. Our analysis predicted that 139 transcripts were differentially represented in the two libraries (p < 0.05), including 72 downregulated and 67 upregulated transcripts. Ninety-five of them matched single UniGene entries (77 known genes and 18 ESTs), while 12 tags corresponded to potentially novel genes. Surprisingly, many of these known genes have never been implicated in chondrogenic differentiation. Interestingly, we found that a significant fraction of these genes formed physical linkage groups. This suggests that the transcriptional control by BMP signaling is in part targeted to genes in certain chromosomal domains. Together, our results provide novel insights into molecular events regulated by BMP signaling in chondrogenesis.

Perivascular adipose tissue (PVAT), an active endocrine organ, exerts direct effect on vascular tone through paracrine. Activation of PVAT metabolism plays an inhibitory role in atherosclerosis via secreting relaxing factors. The present studies were designed to investigate the role of PVAT metabolism in regulation of hypertension.