Bone morphogenetic protein-4 induced by NDRG2 expression inhibits MMP-9 activity in breast cancer cells

In the current study, we examined the function of N-myc downstream-regulated gene 2 (NDRG2) expression in breast cancer cells, especially focusing on the role of bone morphogenetic protein-4 (BMP-4) induced by NDRG2. NDRG2 expression in MDA-MB-231 cells inhibited the mRNA expression of several matrix metalloproteinases (MMPs) and the gelatinolytic activity of MMP-9. Interestingly, a specific induction of active BMP-4 was exclusively observed in MDA-MB-231-NDRG2 cells but not in MDA-MB-231-mock cells. Neutralization of BMP-4 in MDA-MB-231-NDRG2 cells resulted in the rescue of MMP-9 mRNA expression and migration capacity. In addition, treatment with recombinant BMP-4 dramatically suppressed MMP-9 mRNA expression, gelatinolytic MMP-9 activity, migration, and invasion capacity both in MDA-MB-231 and PMA-treated MCF-7 cells. Collectively, our data show that BMP-4 induced by NDRG2 expression inhibits the metastatic potential of breast cancer cells, especially via suppression of MMP-9 activity.     

Bone morphogenetic protein-2 stimulates alkaline phosphatase activity and collagen synthesis in cultured osteoblastic cells, MC3T3-E1

The activities of three bone morphogenetic proteins (BMPs), BMP-1, BMP-2 and BMP-3, on alkaline phosphatase activity, collagen synthesis and DNA synthesis were studied in cultured osteoblastic cells, MC3T3-E1. Treatment of cells with BMP-2 for 48 h induces an increase in cellular alkaline phosphatase activity. This stimulatory effect is evident at a concentration as low as 20 ng/ml of BMP-2 and becomes greater with increasing doses of BMP-2. The BMP-2-induced increase in alkaline phosphatase activity is enhanced by the presence of beta-estradiol, dexamethasone or 1 alpha, 25(OH)2D3. BMP-2 and BMP-3 slightly but significantly stimulate collagen synthesis. None of the BMPs stimulates DNA synthesis in MC3T3-E1 cells at doses tested. These results indicate that BMPs act directly on osteoblastic cells and stimulate the expression of the osteoblastic phenotypes.     

Bone morphogenetic protein-15. Identification of target cells and biological functions

In developing ovarian follicles, the regulation of cell proliferation and differentiation is tightly coordinated. Precisely how this coordination is achieved is unknown, but recent observations have suggested that molecules emitted by the oocyte are involved in the process. The newly discovered oocyte-specific growth factor, bone morphogenetic protein-15 (BMP-15), is one such molecule. At present, nothing is known about the target cells and biological functions of BMP-15. To fill this gap in our knowledge, recombinant BMP-15 and its antibody were produced and used to determine BMP-15 expression and bioactivity. BMP-15 mRNA and protein were shown to be co-expressed in oocytes throughout folliculogenesis, supporting the idea that BMP-15 is a physiological regulator of follicle cell proliferation and/or differentiation. To test this, we used primary cultures of rat granulosa cells (GCs). We found that BMP-15 is a potent stimulator of GC proliferation, and importantly, the mitogenic effect was follicle-stimulating hormone (FSH)-independent. By contrast, BMP-15 alone had no effect on steroidogenesis. However, it produced a marked decrease in FSH-induced progesterone production, but had no effect on FSH-stimulated estradiol production. This result indicates that BMP-15 is a selective modulator of FSH action. In summary, this study identifies GCs as the first target cells for BMP-15. Moreover, it identifies the stimulation of GC proliferation and the differential regulation of two crucial steroid hormones as the first biological functions of BMP-15. Significantly, BMP-15 is the first growth factor that can coordinate GC proliferation and differentiation in a way that reflects normal physiology.     

Bone morphogenetic protein-2 stimulates angiogenesis in developing tumors

Bone Morphogenetic Protein-2 (BMP-2) is highly overexpressed in the majority of patient-derived lung carcinomas. However, a mechanism revealing its role in cancer has not been established. Here we report that BMP-2 enhances the neovascularization of developing tumors. Recombinant BMP-2 stimulated blood vessel formation in tumors formed from A549 cells injected s.c. into thymic nude mice. Recombinant BMP-2 also enhanced angiogenesis in Matrigel plugs containing A549 cells in nude mice. The BMP-2 antagonist noggin abrogated BMP-2-induced angiogenic response. Furthermore, antisense transfection of BMP-2 cDNA resulted in a decrease in blood vessel formation in the Matrigel assays. BMP-2 induced tube formation in both human aortic endothelial cells (HAEC) and umbilical vein endothelial cells. BMP-2 also stimulated proliferation of HAEC. The ability of BMP-2 to activate endothelial cells was further demonstrated by its ability to phosphorylate Smad 1/5/8 and ERK-1/2 and to increase expression of Id1. This study reveals that BMP-2 enhanced the angiogenic response in developing tumors. Furthermore, these data suggest that BMP-2 stimulation of angiogenesis may involve the activation of endothelial cells.     

Angiotensin II stimulates DNA synthesis of rat pancreatic stellate cells by activating ERK through EGF receptor transactivation

Although angiotensin II (Ang II) is known to participate in pancreatic fibrosis, little is known as to the mechanism by which Ang II promotes pancreatic fibrosis. To elucidate the mechanism, we examined the action of Ang II on the proliferation of rat pancreatic stellate cells (PSCs) that play central roles in pancreatic fibrosis. Immunocytochemistry and Western blotting demonstrated that both Ang II type 1 and type 2 receptors were expressed in PSCs. [3H]Thymidine incorporation assay revealed that Ang II enhanced DNA synthesis in PSCs, which was blocked by Ang II type 1 receptor antagonist losartan. Western blotting using anti-phospho-epidermal growth factor (EGF) receptor and anti-phospho-extracellular signal regulated kinase (ERK) antibodies showed that Ang II-activated EGF receptor and ERK. Both EGF receptor kinase inhibitor AG1478 and MEK1 inhibitor PD98059 attenuated ERK activation and DNA synthesis enhanced by Ang II. These results indicate that Ang II stimulates PSC proliferation through EGF receptor transactivation-ERK activation pathway.     

N-acetyl-seryl-aspartyl-lysyl-proline inhibits DNA synthesis in human mesangial cells via up-regulation of cell cycle modulators

N-Acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) was originally reported as a natural inhibitor of the proliferation of stem cells. To elucidate whether Ac-SDKP inhibits the proliferation of human mesangial cells, we examined the effect of Ac-SDKP on fetal calf serum (FCS)- or platelet-derived growth factor (PDGF)-BB-induced DNA synthesis and a cell proliferation. Ac-SDKP inhibited PDGF-BB- or FCS-induced DNA synthesis without cellular toxicity. The protein expression of p53 and p27kip1 was significantly increased by Ac-SDKP. Ac-SDKP also up-regulated the PDGF-BB-stimulated expression of p21cip1 and suppressed PDGF-BB-induced cyclin D1 expression. In p53 knock-out human mesangial cells made with small interference RNA, the protein expression of p21cip1 and p27kip1 was also decreased and the inhibitory effect of Ac-SDKP on mesangial proliferation was completely abolished. Ac-SDKP increased the stability of p53 protein as demonstrated by pulse-chase experiment. These results suggest that p53 is the key mediator of Ac-SDKP-induced inhibition of DNA synthesis through the up-regulation of cell cycle modulators, highlighting a potential effect of Ac-SDKP on various progressive renal diseases.     

Matrix GLA protein modulates differentiation induced by bone morphogenetic protein-2 in C3H10T1/2 cells

Matrix GLA protein (MGP) is ubiquitously expressed with high accumulation in bone and cartilage, where it was found to associate with bone morphogenetic proteins (BMP) during protein purification. To test whether MGP affects BMP-induced differentiation, three sets of experiments were performed. First, pluripotent C3H10T1/2 cells transfected with human MPG (hMGP) or antisense to hMGP (AS-hMGP) were treated with BMP-2. In cells overexpressing hMGP, osteogenic and chondrogenic differentiation was inhibited indicating decreased BMP-2 activity. Conversely, in cells overexpressing AS-hMGP, BMP-2 activity was enhanced. Second, cells were prepared from homozygous and heterozygous MPG-deficient mice aortas. When treated with BMP-2, these cells underwent chondrogenic and osteogenic differentiation, respectively, whereas controls did not. Third, FLAG-tagged hMGP with the same biological effect as native hMGP inhibited BMP-induced differentiation, when exogenously added to culture media. Together, these results suggest that MGP modulates BMP activity. To test whether hMGP fragments would retain the effect of full-length hMGP, three subdomains were overexpressed in C3H10T1/2 cells. In cells expressing the mid-region, alone (amino acids (aa) 35-54) or in combination with the N terminus (aa 1-54) but not the C terminus (aa 35-84), osteogenic differentiation was enhanced and occurred even without added BMP-2. Thus, two subdomains had the opposite effect of full-length hMGP, possibly due to different expression levels or domain characteristics.     

Hypertrophy is induced during the in vitro chondrogenic differentiation of human mesenchymal stem cells by bone morphogenetic protein-2 and bone morphogenetic protein-4 gene transfer

Introduction  

The present study compares bone morphogenetic protein (BMP)-4 and BMP-2 gene transfer as agents of chondrogenesis and hypertrophy in human primary mesenchymal stem cells (MSCs) maintained as pellet cultures.     

Signal transductions induced by bone morphogenetic protein-2 and transforming growth factor-beta in normal human osteoblastic cells

Transforming growth factor beta (TGF-beta) activates Ras/MAPK signaling in many cell types. Because TGF-beta and BMP-2 exert similar effects, we examined if this signaling is stimulated by both factors and analyzed the relationship between this signaling and the Smads in osteoblasts. BMP-2 and TGF-beta stimulated Ras, MAPK, and AP-1 activities. The DNA binding activities of c-Fos, FosB/Delta FosB, Fra-1, Fra-2, and JunB were up-regulated whereas JunD activity was decreased. c-Fos, FosB/Delta FosB, and JunB were associated with Smad4. The stimulation of AP-1 by BMP-2 and TGF-beta was dependent on Smad signaling, and anti-Smad4 antibody interfered with AP-1 activity. Thus, BMP-2 and TGF-beta activate both Ras/MAPK/AP-1 and Smad signaling in osteoblasts with Smads modulating AP-1 activity. To determine the roles of MAPK in BMP-2 and TGF-beta function, we analyzed the effect of ERK and p38 inhibitors on the regulation of bone matrix protein expression and JunB and JunD levels by these two factors. ERK and p38 mediated TGF-beta suppression of osteocalcin and JunD as well as stimulation of JunB. p38 was essential in BMP-2 up-regulation of type I collagen, fibronectin, osteopontin, osteocalcin, and alkaline phosphatase activity whereas ERK mediated BMP-2 stimulation of fibronectin and osteopontin. Thus, ERK and p38 differentially mediate TGF-beta and BMP-2 function in osteoblasts.     

Cytokine-induced production of monocyte chemoattractant protein-1 by cultured human mesangial cells.

The infiltration of the glomerulus by monocyte-derived macrophages is an important step in the pathogenesis of glomerular injury. The factors regulating glomerular leukocyte traffic remain unknown. We postulated that the glomerular mesangial cell (MC) may participate in the development of glomerular inflammation through the production of the monocyte-specific chemotactic factor, monocyte chemoattractant protein-1 (MCP-1). Using a cell culture system, we found that human MC produced a basal level of monocyte chemotactic activity, which was significantly increased by the inflammatory cytokines IL-1 beta and TNF-alpha. This increase in bioactivity correlated with the increased expression of MCP-1 mRNA by cytokine-conditioned MC. The total chemotactic activity of MC-conditioned supernatants was reduced by more than 80% after immunoadsorption with a specific anti-MCP-1 antibody. Thus, MC could play a role in inflammatory glomerular conditions through the production of MCP-1.     

Cd++ inhibits EGF-induced DNA synthesis but not EGF induced myc mRNA accumulation in serum starved NRK-49F cells

Cd⁺⁺ inhibits EGF-induced ³H-thymidine incorporation in serum deprived NRK-49F cells in a dose dependent pattern. The underlying mechanisms for this inhibition are largely unknown. EGF-induced myc mRNA accumulation in NRK-49F cells and the effects of Cd⁺⁺ on this response were examined under conditions that result in partial or complete inhibition of EGF-induced DNA synthesis. It was found that doses of Cd⁺⁺ that inhibit EGF-induced DNA synthesis do not inhibit EGF-induced protein synthesis and myc mRNA accumulation. Cd⁺⁺ doses of 0.5 µM and 1 µM were found actually to increase EGF-induced myc mRNA accumulation and amino acid incorporation. These results show that the effect of Cd⁺⁺ on EGF-induced DNA synthesis is not due to inhibition of entrance into G₁, but rather that Cd⁺⁺ acts on events subsequent to myc accumulation; that is, events associated with either G₁ progression, entry into S or DNA synthesis.Abbreviations EGF Epidermal Growth Factor - ³HTdr Tritium thymidine - MeAIB Methylaminoisobutyrate     

ERK phosphorylation potentiates Elk-1-mediated ternary complex formation and transactivation.

Induction of the human c-fos proto-oncogene by mitogens depends on the formation of a ternary complex by p62TCF with the serum response factor (SRF) and the serum response element (SRE). We demonstrate that Elk-1, a protein closely related to p62TCF in function, is a nuclear target of two members of the MAP kinase family, ERK1 and ERK2. Phosphorylation of Elk-1 increases the yield of ternary complex in vitro. At least five residues in the C-terminal domain of Elk-1 are phosphorylated upon growth factor stimulation of NIH3T3 cells. These residues are also phosphorylated by purified ERK1 in vitro, as determined by a combination of phosphopeptide sequencing and 2-D peptide mapping. Conversion of two of these phospho-acceptor sites to alanine impairs the formation of ternary complexes by the resulting Elk-1 proteins. Removal of these serine residues also drastically diminishes activation of the c-fos promoter in epidermal growth factor-treated cells. Analogous mutations at other sites impair activation to a lesser extent without affecting ternary complex formation in vitro. Our results indicate that phosphorylation regulates ternary complex formation by Elk-1, which is a prerequisite for the manifestation of its transactivation potential at the c-fos SRE.     

Bone morphogenetic protein-4 abrogates lumen formation by mammary epithelial cells and promotes invasive growth

Bone morphogenetic proteins (BMPs) are multifunctional cytokines that regulate key developmental processes, but are also overexpressed in many carcinomas. To assess whether BMPs would influence the three-dimensional architecture of epithelial structures, we took advantage of an in vitro model in which mammary epithelial cells form alveolar-like spherical cysts in collagen gels. We found that BMP-4 has a dramatic, biphasic effect on the organization of epithelial cysts. When added in the concentration range of 1-10 ng/ml, the cytokine abrogates lumen formation and induces the outgrowth of multiple invasive cord-like structures. At higher concentrations (20-100 ng/ml), BMP-4 additionally disrupts cell-cell adhesion, resulting in cyst disintegration and scattering of individual cells into the surrounding collagen matrix. The finding that BMP-4 subverts the ability of mammary epithelial cells to form polarized lumen-containing structures and endows them with invasive properties supports the involvement of this cytokine in the progression of breast cancer.