06-702 Sigma-AldrichAnti-phospho-Smad1 (Ser463/465) Antibody

Transforming growth factor β (TGF-β) and related cytokines play a central role in the vascular system. In vitro, TGF-β induces aortic endothelial cells to assemble subcellular actin-rich structures specialized for matrix degradation called podosomes. To explore further this TGF-β-specific response and determine in which context podosomes form, ALK5 and ALK1 TGF-β receptor signaling pathways were investigated in bovine aortic endothelial cells. We report that TGF-β drives podosome formation through ALK5 and the downstream effectors Smad2 and Smad3. Concurrent TGF-β-induced ALK1 signaling mitigates ALK5 responses through Smad1. ALK1 signaling induced by BMP9 also antagonizes TGF-β-induced podosome formation, but this occurs through both Smad1 and Smad5. Whereas ALK1 neutralization brings ALK5 signals to full potency for TGF-β-induced podosome formation, ALK1 depletion leads to cell disturbances not compatible with podosome assembly. Thus, ALK1 possesses passive and active modalities. Altogether, our results reveal specific features of ALK1 and ALK5 signaling with potential clinical implications.

Heart failure is a major cause of morbidity and mortality worldwide. The ubiquitously expressed cytokine transforming growth factor-β1 (TGFβ1) promotes cardiac fibrosis, an important component of progressive heart failure. Membrane-associated endoglin is a coreceptor for TGFβ1 signaling and has been studied in vascular remodeling and preeclampsia. We hypothesized that reduced endoglin expression may limit cardiac fibrosis in heart failure.We first report that endoglin expression is increased in the left ventricle of human subjects with heart failure and determined that endoglin is required for TGFβ1 signaling in human cardiac fibroblasts using neutralizing antibodies and an siRNA approach. We further identified that reduced endoglin expression attenuates cardiac fibrosis, preserves left ventricular function, and improves survival in a mouse model of pressure-overload-induced heart failure. Prior studies have shown that the extracellular domain of endoglin can be cleaved and released into the circulation as soluble endoglin, which disrupts TGFβ1 signaling in endothelium. We now demonstrate that soluble endoglin limits TGFβ1 signaling and type I collagen synthesis in cardiac fibroblasts and further show that soluble endoglin treatment attenuates cardiac fibrosis in an in vivo model of heart failure.Our results identify endoglin as a critical component of TGFβ1 signaling in the cardiac fibroblast and show that targeting endoglin attenuates cardiac fibrosis, thereby providing a potentially novel therapeutic approach for individuals with heart failure.

Members of the bone morphogenetic proteins (BMPs) superfamily are expressed in the testis and epididymis and are believed to have different biological functions during testicular and epididymal development. Smad1 is one of the signal transducers of BMP signaling and binds to several proteins involved in ubiquitin-proteasome system (UPS). Valosin-containing protein (p97/VCP) is required for the degradation of some UPS substrates. Although p97/VCP has been indicated in different cellular pathways, its association with BMP signaling in male reproductive system has not been elucidated. The aim of the present study was to investigate the cellular localization of Smad1, phospho-Smad1, and p97/VCP and the interaction of proteins in the postnatal rat testis and epididymis. Testicular and epididymal tissues from 5-, 15- and 60-day-old rats were examined by immunohistochemistry, immunofluorescence, Western blotting, and immunoprecipitation techniques. In 5-day-old rat testis, Smad1, phospho-Smad1, and p97/VCP were mainly expressed in gonocytes. In 15- and 60-day-old rat testis, proteins were overlapped in spermatogonia, Sertoli cells, and spermatocytes. Expression of proteins in the epithelial cells of epididymis was gradually increased from 5 to 15 days of age. Smad1 and phospho-Smad1 expressions showed uniformity in the different regions of epididymis, however p97/VCP immunoreactivity was higher only in caput epididymis compared to corpus and cauda epididymis in 15- and 60-day-old rat epididymis. Co-immunoprecipitation experiments further confirmed the Smad1-p97/VCP and p-Smad1-p97/VCP interactions. The overlap between Smad1 and p97/VCP expressions in the postnatal rat testis and epididymis suggests that p97/VCP may play important roles in mediating BMP signaling during spermatogenesis.

The bone morphogenetic protein (BMP) family is a class of transforming growth factor (TGF-beta) superfamily molecules that have been implicated in neuronal differentiation. We studied the effects of BMP2 and glial cell line-derived neurotrophic factor (GDNF) on inducing differentiation of enteric neurons and the signal transduction pathways involved. Studies were performed using a novel murine fetal enteric neuronal cell line (IM-FEN) and primary enteric neurons. Enteric neurons were cultured in the presence of vehicle, GDNF (100 ng/ml), BMP2 (10 ng/ml), or both (GDNF + BMP2), and differentiation was assessed by neurite length, markers of neuronal differentiation (neurofilament medium polypeptide and beta-III-tubulin), and neurotransmitter expression [neuropeptide Y (NPY), neuronal nitric oxide synthase (nNOS), tyrosine hydroxylase (TH), choline acetyltransferase (ChAT) and Substance P]. BMP2 increased the differentiation of enteric neurons compared with vehicle and GDNF-treated neurons (P < 0.001). BMP2 increased the expression of the mature neuronal markers (P < 0.05). BMP2 promoted differentiation of NPY-, nNOS-, and TH-expressing neurons (P < 0.001) but had no effect on the expression of cholinergic neurons (ChAT, Substance P). Neurons cultured in the presence of BMP2 have higher numbers of TH-expressing neurons after exposure to 1-methyl 4-phenylpyridinium (MPP(+)) compared with those cultured with MPP(+) alone (P < 0.01). The Smad signal transduction pathway has been implicated in TGF-beta signaling. BMP2 induced phosphorylation of Smad1, and the effects of BMP2 on differentiation of enteric neurons were significantly reduced in the presence of Smad1 siRNA, implicating the role of Smad1 in BMP2-induced differentiation. The effects of BMP2 on catecholaminergic neurons may have therapeutic implications in gastrointestinal motility disturbances.

The bone morphogenetic protein (BMP) family represents a class of signaling molecules, that plays key roles in morphogenesis, cell proliferation, survival and differentiation during normal development. Members of this family are essential for the development of the mammalian orofacial region where they regulate cell proliferation, extracellular matrix synthesis, and cellular differentiation. Perturbation of any of these processes results in orofacial clefting. Embryonic orofacial tissue expresses BMP mRNAs, their cognate proteins, and BMP-specific receptors in unique temporo-spatial patterns, suggesting functional roles in orofacial development. However, specific genes that function as downstream mediators of BMP action during orofacial ontogenesis have not been well defined. In the current study, elements of the Smad component of the BMP intracellular signaling system were identified and characterized in embryonic orofacial tissue and functional activation of the Smad pathway by BMP2 and BMP4 was demonstrated. BMP2 and BMP4-initiated Smad signaling in cells derived from embryonic orofacial tissue was found to result in: (1) phosphorylation of Smads 1 and 5; (2) nuclear translocation of Smads 1, 4, and 5; (3) binding of Smads 1, 4, and 5 to a consensus Smad binding element (SBE)-containing oligonucleotide; (4) transactivation of transfected reporter constructs, containing BMP-inducible Smad response elements; and (5) increased expression at transcriptional as well as translational levels of Id3 (endogenous gene containing BMP receptor-specific Smad response elements). Collectively, these data document the existence of a functional Smad-mediated BMP signaling system in cells of the developing murine orofacial region.

It is widely accepted that vitreous humor-derived FGFs are required for the differentiation of anterior lens epithelial cells into crystallin-rich fibers. We show that BMP2, 4, and 7 can induce the expression of markers of fiber differentiation in primary lens cell cultures to an extent equivalent to FGF or medium conditioned by intact vitreous bodies (VBCM). Abolishing BMP2/4/7 signaling with noggin inhibited VBCM from upregulating fiber marker expression. Remarkably, noggin and anti-BMP antibodies also prevented purified FGF (but not unrelated stimuli) from upregulating the same fiber-specific proteins. This effect is attributable to inhibition of BMPs produced by the lens cells themselves. Although BMP signaling is required for FGF to enhance fiber differentiation, the converse is not true. Expression of noggin in the lenses of transgenic mice resulted in a postnatal block of epithelial-to-secondary fiber differentiation, with extension of the epithelial monolayer to the posterior pole of the organ. These results reveal the central importance of BMP in secondary fiber formation and show that although FGF may be necessary for this process, it is not sufficient. Differentiation of fiber cells, and thus proper vision, is dependent on cross-talk between the FGF and BMP signaling pathways.

Homeostasis in the lens is dependent on an extensive network of cell-to-cell gap junctional channels. Gap junction-mediated intercellular coupling (GJIC) is higher in the equatorial region of the lens than at either pole, an asymmetry believed essential for lens transparency. Primary cultures of embryonic chick lens epithelial cells up-regulate GJIC in response to purified fibroblast growth factor (FGF)1/2 or to medium conditioned by vitreous bodies, the major reservoir of factors (including FGF) for the lens equator. We show that purified bone morphogenetic protein (BMP)2, -4, and -7 also up-regulate GJIC in these cultures. BMP2, -4, or both are present in vitreous body conditioned medium, and BMP4 and -7 are endogenously expressed by lens cells. Remarkably, lens-derived BMP signaling is required for up-regulation of GJIC by purified FGF, and sufficient for up-regulation by vitreous humor. This is the first demonstration of an obligatory interaction between FGF and BMPs in postplacode lens cells, and of a role for FGF/BMP cross-talk in regulating GJIC in any cell type. Our results support a model in which the angular gradient in GJIC in the lens, and thus proper lens function, is dependent on signaling between the FGF and BMP pathways.

Astrocytes play important roles in brain development and injury response. Transcription factors STAT3 and Smad1, activated by leukemia inhibitory factor (LIF) and bone morphogenetic protein 2 (BMP2), respectively, form a complex with the coactivator p300 to synergistically induce astrocytes from neuroepithelial cells (NECs) (K. Nakashima, M. Yanagisawa, H. Arakawa, N. Kimura, T. Hisatsune, M. Kawabata, K. Miyazono, and T. Taga, Science 284:479-482, 1999). However, the mechanisms that govern astrogliogenesis during the determination of the fate of neural stem cells remain elusive. Here we found that LIF induces expression of BMP2 via STAT3 activation and leads to the consequent activation of Smad1 to efficiently promote astrogliogenic differentiation of NECs. The BMP antagonist Noggin abrogated LIF-induced Smad1 activation and astrogliogenesis by inhibiting BMPs produced by NECs. NECs deficient in suppressor of cytokine signaling 3 (SOCS3), a negative regulator of STAT3, readily differentiated into astrocytes upon activation by LIF not only due to sustained activation of STAT3 but also because of the consequent activation of Smad1. Our study suggests a novel LIF-triggered positive regulatory loop that enhances astrogliogenesis.

Bone morphogenetic proteins (BMPs) are members of the transforming growth factor-beta superfamily, which utilize BMP receptors and intracellular SMADs to transduce their signals to regulate cell differentiation, proliferation, and apoptosis. Because mutations in BMP receptor type IA (BMPRIA) and SMAD4 are found in the germline of patients with the colon cancer predisposition syndrome juvenile polyposis, and because the contribution of BMP in colon cancers is largely unknown, we examined colon cancer cells and tissues for evidence of BMP signaling and determined its growth effects. We determined the presence and functionality of BMPR1A by examining BMP-induced phosphorylation and nuclear translocation of SMAD1; transcriptional activity via a BMP-specific luciferase reporter; and growth characteristics by cell cycle analysis, cell growth, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide metabolic assays. These assays were also performed after transfection with a dominant negative (DN) BMPR1A construct. In SMAD4-null SW480 cells, we examined BMP effects on cellular wound assays as well as BMP-induced transcription in the presence of transfected SMAD4. We also determined the expression of BMPR1A, BMP ligands, and phospho-SMAD1 in primary human colon cancer specimens. We found intact BMP signaling and modest growth suppression in HCT116 and two derivative cell lines and, surprisingly, growth suppression in SMAD4-null SW480 cells. BMP-induced SMAD signaling and BMPR1A-mediated growth suppression were reversed with DN BMPR1A transfection. BMP2 slowed wound closure, and transfection of SMAD4 into SW480 cells did not change BMP-specific transcriptional activity over controls due to receptor stimulation by endogenously produced ligand. We found no cell cycle alterations with BMP treatment in the HCT116 and derivative cell lines, but there was an increased G1 fraction in SW480 cells that was not due to increased p21 transcription. In human colon cancer specimens, BMP2 and BMP7 ligands, BMPRIA, and phospho-SMAD1 were expressed. In conclusion, BMP signaling is intact and growth suppressive in human colon cancer cells. In addition to SMADs, BMP may utilize SMAD4-independent pathways for growth suppression in colon cancers.

We show that 1 of the type II bone morphogenetic protein (BMP) receptor ligands, BMP4, is widely expressed in the adult mouse lung and is upregulated in hypoxia-induced pulmonary hypertension (PH). Furthermore, heterozygous null Bmp4(lacZ/+) mice are protected from the development of hypoxia-induced PH, vascular smooth muscle cell proliferation, and vascular remodeling. This is associated with a reduction in hypoxia-induced Smad1/5/8 phosphorylation and Id1 expression in the pulmonary vasculature. In addition, pulmonary microvascular endothelial cells secrete BMP4 in response to hypoxia and promote proliferation and migration of vascular smooth muscle cells in a BMP4-dependent fashion. These findings indicate that BMP4 plays a dominant role in regulating BMP signaling in the hypoxic pulmonary vasculature and suggest that endothelium-derived BMP4 plays a direct, paracrine role in promoting smooth muscle proliferation and remodeling in hypoxic PH.