Sherry L. Abboud

Requirement of BMP-2-induced phosphatidylinositol 3-kinase and Akt serine/threonine kinase in osteoblast differentiation and Smad-dependent BMP-2 gene transcription.

The mechanism by which bone morphogenetic protein-2 (BMP-2) induces osteoblast differentiation is not precisely known. We investigated the involvement of the phosphatidylinositol (PI) 3-kinase/Akt signal transduction pathway in modulation of this process. BMP-2 stimulated PI 3-kinase activity in osteogenic cells. Inhibition of PI 3-kinase activity with the specific inhibitor Ly-294002 prevented BMP-2-induced alkaline phosphatase, an early marker of osteoblast differentiation. Expression of dominant-negative PI 3-kinase also abolished osteoblastic induction of alkaline phosphatase in response to BMP-2, confirming the involvement of this lipid kinase in this process. BMP-2 stimulated Akt serine/threonine kinase activity in a PI 3-kinase-dependent manner in osteoblast precursor cells. Inhibition of Akt activity by a dominant-negative mutant of Akt blocked BMP-2-induced osteoblastic alkaline phosphatase activity. BMP-2 stimulates its own expression during osteoblast differentiation. Expression of dominant-negative PI 3-kinase or dominant-negative Akt inhibited BMP-2-induced BMP-2 transcription. Because all the known biological activities of BMP-2 are mediated by transcription via BMP-specific Smad proteins, we investigated the involvement of PI 3-kinase in Smad-dependent BMP-2 transcription. Smad5 stimulated BMP-2 transcription independent of addition of the ligand. Dominant-negative PI 3-kinase or dominant-negative Akt inhibited Smad5-dependent transcription of BMP-2. Furthermore dominant-negative Akt inhibited translocation of BMP-specific Smads into nucleus. Together these data provide the first evidence that activation of BMP receptor serine/threonine kinase stimulates the PI 3 kinase/Akt pathway and define a role for this signal transduction pathway in BMP-specific Smad function during osteoblast differentiation.

Secretion of insulinlike growth factor I and insulinlike growth factor-binding proteins by murine bone marrow stromal cells.

Insulin-like growth factor I (IGF-I) stimulates hematopoiesis. We examined whether bone marrow stromal cells synthesize IGF-I. Secretion of IGF-I immunoreactivity by cells from TC-1 murine bone marrow stromal cells was time-dependent and inhibited by cycloheximide. Gel filtration chromatography under denaturing conditions of TC-1 conditioned medium demonstrated two major peaks of apparent IGF-I immunoreactivity with molecular weights of approximately 7.5-8.0 kD, the size of native IGF-I, and greater than 25 kD. Expression of IGF-I mRNA was identified by both RNase protection assay and reverse transcription/polymerase chain reaction. To determine whether the greater than 25-kD species identified by RIA possessed IGF-binding activity, a potential cause of artifactual IGF-I immunoreactivity, charcoal adsorption assay of these gel filtration fractions was performed. The peak of IGF-binding activity coeluted with apparent IGF-I immunoreactivity suggesting that TC-1 cells secrete IGF-binding protein(s). Unfractionated conditioned medium exhibited linear dose-dependent increase in specific binding of [125I]-IGF-I with a pattern of displacement (IGF-I and IGF-II much greater than insulin) characteristic of IGF-binding proteins. Western ligand analysis of conditioned medium showed three IGF-I binding species of approximately 31, 38, and 40 kD. These data indicate that TC-1 bone marrow stromal cells synthesize and secrete IGF-I and IGF-binding proteins and constitute a useful model system to study their regulation and role in hematopoiesis.

Bone morphogenetic protein-2 induces cyclin kinase inhibitor p21 and hypophosphorylation of retinoblastoma protein in estradiol-treated MCF-7 human breast cancer cells

The biologic effects and mechanisms by which bone morphogenetic proteins (BMPs) function in breast cancer cells are not well defined. A member of this family of growth and differentiation factors, BMP-2, inhibited both basal and estradiol-induced growth of MCF-7 breast tumor cells in culture. Flow cytometric analysis showed that in the presence of BMP-2, 62% and 45% of estradiol-stimulated MCF-7 cells progressed to S-phase at 24 h and 48 h, respectively. Estradiol mediates growth of human breast cancer cells by stimulating cyclins and cyclin-dependent kinases (CDKs). BMP-2 significantly increased the level of the cyclin kinase inhibitor, p21, which in turn associated with and inactivated cyclin D1. BMP-2 inhibited estradiol-induced cyclin D1-associated kinase activity. Also estradiol-induced CDK2 activity was inhibited by BMP-2. This inhibition of CDK activity resulted in hypophosphorylation of retinoblastoma protein thus keeping it in its active form. These data provide the first evidence by which BMP-2 inhibits estradiol-induced proliferation of human breast cancer cells.

Characterization and regulation of insulin-like growth factor binding proteins in human hepatic stellate cells.

Cultured hepatic stellate cells (HSCs), the cell type primarily involved in the progression of liver fibrosis, secrete insulin‐like growth factor‐I (IGF‐I) and IGF binding protein (IGFBP) activity. IGF‐I exerts a mitogenic effect on HSCs, thus potentially contributing to the fibrogenic process in an autocrine fashion. However, IGF‐I action is modulated by the presence of specific IGFBPs that may inhibit and/or enhance its biologic effects. Therefore, we examined IGFBP‐1 through IGFBP‐6 mRNA and protein expression in HSCs isolated from human liver and activated in culture. Regulation of IGFBPs in response to IGF‐I and other polypeptide growth factors involved in the hepatic fibrogenic process was also assessed. RNase protection assays and ligand blot analysis demonstrated that HSCs express IGFBP‐2 through IGFBP‐6 mRNAs and release detectable levels of IGFBP‐2 through IGFBP‐5. Because IGF‐I, platelet‐derived growth factor‐BB (PDGF‐BB), and transforming growth factor‐β (TGF‐β) stimulate HSC proliferation and/or matrix production, we tested their effect on IGFBPs released by HSCs. IGF‐I induced IGFBP‐3 and IGFBP‐5 proteins in a time‐dependent manner without an increase in the corresponding mRNAs. IGFBP‐4 protein levels decreased in response to IGF‐I. TGF‐β stimulated IGFBP‐3 mRNA and protein but decreased IGFBP‐5 mRNA and protein. In contrast, PDGF‐BB failed to regulate IGFBPs compared with controls. Recombinant human IGFBP‐3 (rhIGFBP‐3) was then tested for its effect on IGF‐I‐induced mitogenesis in HSCs. rhIGFBP‐3 inhibited IGF‐I‐stimulated DNA synthesis in a dose‐dependent manner, with a peak effect observed at 25 nM IGFBP‐3. Because TGF‐β is highly expressed in cirrhotic liver tissue, we determined whether IGFBP‐3 mRNA expression is increased in liver biopsies obtained from patients with an active fibroproliferative response due to viral‐induced chronic active hepatitis. In the majority of these samples, IGFBP‐3 mRNA was increased compared with normal controls. These findings indicate that human HSCs, in their activated phenotype, constitutively produce IGFBPs. IGF‐I and TGF‐β differentially regulate IGFBP‐3, IGFBP‐4, and IGFBP‐5 expression, which, in turn, may modulate the in vitro and in vivo action of IGF‐I. J. Cell. Physiol. 174:240–250, 1998.

BMP-2 regulates cardiomyocyte contractility in a phosphatidylinositol 3 kinase-dependent manner.

Bone morphogenetic protein‐2 (BMP‐2) regulates development of heart during vertebrate embryogenesis. In vitro BMP‐2 induces differentiation of precardiac cells into mature cardiomyocytes by inducing the expression of cardiac‐specific genes. However, the role of BMP‐2 and its signaling in other cardiac functions have not been studied. We examined the action of phosphatidylinositol (PI) 3 kinase in isolated adult rat cardiomyocytes. Incubation of rat ventricular cardiomyocytes with BMP‐2 increased the PI 3 kinase activity. Ly294002, a pharmacological inhibitor of PI 3 kinase, blocked BMP‐2‐induced PI 3 kinase activity completely. To investigate the contractility of isolated cardiomyocytes, fractional shortening was examined. BMP‐2 significantly increased the percent fractional shortening of the cardiomyocytes. Inhibition of PI 3 kinase activity completely abolished this action of BMP‐2. These data indicate that PI 3 kinase regulates BMP‐2‐induced myocyte contractility. To further confirm this observation, we used adenovirus‐mediated gene transfer to express a constitutively active myristoylated catalytic subunit of PI 3 kinase in rat cardiomyocytes. Infection of cardiomyocytes with the adenovirus vector increased the expression of constitutively active PI 3 kinase within 24 h. Expression of constitutively active PI 3 kinase significantly increased cardiomyocyte contractility. Together, these data show for the first time that the growth and differentiation factor, BMP‐2, stimulates cardiomyocyte contractility. Also we provide the first evidence that BMP‐2‐induced PI 3 kinase activity regulates this cardiomyocyte function.

Characterization of insulin-like growth factor binding proteins (IGFBP) and regulation of IGFBP-4 in bone marrow stromal cells

Summary. Bone marrow stromal cells synthesize and secrete insulin‐like growth factor (IGF)‐I and IGF‐binding proteins (IGFBP). IGFBPs may modulate the action of IGF‐I or IGF‐II on haemopoiesis. However, the specific IGFBPs produced by various stromal cell types have not been identified. We examined six different stromal phenotypes for IGFBP protein and IGFBP‐1 to ‐6 mRNA expression. [125I]IGF‐I ligand blot analysis of conditioned medium demonstrate different patterns of IGFBP secretion by each cell type. The most prominent IGFBPs were 24 and 29 kD species, consistent with IGFBP4 and IGFBP5, respectively. RNase protection assays demonstrate that, overall, stromal cells express IGFBP‐2 to ‐6 mRNAs, with IGFBP4, IGFBP5 and IGFBP6 mRNAs predominating. Since agents that modulate cAMP levels may influence haemopoiesis via the release of stromal‐derived cytokines, we determined the effect of forskolin, a cAMP agonist, on IGFBP4 expression in TC‐1 cells. Forskolin (10 5 M) up‐regulated IGFBP4 mRNA and protein secretion in a time‐dependent manner. These findings suggest that IGFBP‐4, ‐5 and ‐6 released by stromal cells may be key modulators of the haemopoietic response to IGFs. Release of IGFBP4 by agents that increase cAMP may be an important mechanism involved in regulating IGF bioavailability in the marrow microenvironment.

Preferential expression of insulin-like growth factor binding proteins- 1, -3, and -5 during early diabetic renal hypertrophy in rats

The renal insulin-like growth factor-I (IGF-I) system has been implicated in the pathogenesis of renal hypertrophy, altered hemodynamics, and extracellular matrix expansion associated with early diabetes. The relative abundance of IGF binding proteins (IGFBPs) in the renal microenvironment may modulate IGF-I actions. However, the precise IGFBPs expressed in the glomerular and tubulointerstitial compartments during diabetic renal growth have not been characterized. In the present study, in situ hybridization studies were performed to examine the expression of IGFBP-1 to -6 messenger RNAs (mRNAs) 3, 7, and 14 days after streptozotocin (STZ) injection in rats. In control, nondiabetic kidneys, all six IGFBP mRNAs were differentially expressed with a predominance of IGFBP-5. The onset of renal hypertrophy in STZ-induced diabetes was associated with a rapid and site-specific induction of IGFBP-1, -3, and -5 mRNAs. In contrast, basal expression of IGFBP-2, -4, and -6 mRNAs was not altered in diabetic rats. IGFBP-5 mRNA expression increased in diabetic glomeruli, cortical, and inner medullary peritubular interstitial cells at days 3, 7, and 14. Although normal glomeruli failed to express IGFBP-3, it was induced concomitantly with IGFBP-5 in diabetic glomeruli and cortical peritubular interstitial cells. IGFBP-1 mRNA levels also increased in cortical tubular cells at each time point tested. Peak induction of IGFBP-3 and -5 was observed at day 3, whereas IGFBP-1 was delayed until day 7. IGFBP-1, -3, and -5 mRNA levels declined by day 14, but remained persistently elevated above control. By immunoperoxidase staining, similar alterations in the pattern of IGFBP-3 and -5 protein expression were observed at each time point. The preferential and site-specific increase in IGFBP-1, -3, and -5 suggest that these IGFBPs may regulate the local autocrine and/or paracrine actions of IGF-I and contribute to the pathogenesis of the early manifestations of diabetic nephropathy.

Regulation of basement membrane heparan sulfate proteoglycan, perlecan, gene expression in glomerular epithelial cells by high glucose medium

Proteinuria in diabetic nephropathy has been correlated with reduction in heparan sulfate proteoglycan (HSPG) content of the glomerular basement membrane. We have previously shown that the underlying mechanism probably involves reduction in the synthesis by glomerular epithelial cells. In this study we explored whether high glucose medium regulates basement membrane HSPG gene expression. Northern analysis demonstrated that rat glomerular epithelial cells in vitro constitutively express mRNA for basement membrane HSPG, similar to that observed in rat kidney glomerulus. RNase protection assay showed that incubation of glomerular epithelial cells with 30 mM glucose for 24 h and 7 days resulted in reduction in HSPG mRNA abundance. The decrease in mRNA abundance correlated with reduction in the synthesis of 35 SO4‐labeled basement membrane HSPG as measured by immunoprecipitation. Reduction in synthesis of HSPG could not be entirely accounted for by decrease in mRNA abundance, suggesting both transcriptional and posttranscriptional mechanisms may be involved in reduction of glomerular basement membrane HSPG synthesis by glomerular epithelial cells in diabetic nephropathy.

Potential role of insulin‐like growth factor binding protein‐4 in the uncoupling of bone turnover in multiple myeloma

Decreased bone formation plays an important role in the development of lytic lesions during the late stage of multiple myeloma (MM). Release of insulin‐like growth factor binding protein‐4 (IGFBP4) by tumour cells adjacent to bone may inhibit IGF‐I‐stimulated osteoblast growth and contribute to decreased bone formation. The present study demonstrates that the human MM cell line, ARH‐77, expresses IGFBP4 and, to a lesser extent, IGFBP6 mRNA and protein. IGFBP4 expression in myeloma cells may be modulated by cytokines released by stromal cells and T cells in the microenvironment. We tested the effect of recombinant interferon‐gamma (INF) on IGFBP4 expression in ARH‐77. INF increased IGFBP4 mRNA and protein levels at 12 h, with a decline to baseline by 24 h. In contrast, IGFBP4 was not regulated in response to IL‐6, TNF‐alpha, PDGF BB, bFGF, TGF‐beta or the cAMP agonist, forskolin. In other systems, IGFBP4 may also be regulated post‐transcriptionally by a protease that is activated by IGF‐I or ‐II. Conditioned medium from ARH‐77 cultures incubated with IGF‐I or ‐II for up to 24 h failed to demonstrate proteolytic activity. Proteolysis was also not observed when conditioned medium containing exogenous rhIGFBP4 was incubated with IGF‐I or ‐II under cell‐free conditions. To determine if human myeloma tumours also express IGFBP4, total RNA was isolated from four tumour biopsies. All samples expressed detectable levels of IGFBP4 mRNA. These findings indicate that interferon‐gamma may indirectly modulate bone formation via the the release of tumour‐derived IGFBP4, suggesting that the immune system may influence bone turnover in MM. Failure of myeloma cells to release protease activity may promote IGFBP4 accumulation in the microenvironment during tumour growth.

PDGF BB and bFGF stimulate DNA synthesis and upregulate CSF-1 and MCP-1 gene expression in dental follicle cells

CSF-1 and MCP-1, released by dental follicle cells, stimulate the influx of monocytes into the follicle sac and enhance the formation of osteoclasts that, in turn, resorb alveolar bone for the eruption pathway. PDGF and bFGF, released by cells adjacent to the follicle or by activated monocytes, are prime candidates that may regulate CSF-1 and MCP-1 gene expression. The present study demonstrates that PDGF and bFGF are mitogens for dental follicle cells and stimulate CSF-1 and MCP-1 mRNA, but with different time course kinetics. Peak induction of CSF-1 mRNA was observed at 6-8h, while maximal MCP-1 induction was observed at 2h. These findings suggest that MCP-1 is an early chemotactic signal for monocytes and that subsequent release of CSF-1 may act synergistically with MCP-1 to enhance monocyte influx. Further understanding of the molecular mechanisms by which cytokines regulate CSF-1 and MCP-1 may lead to more effective treatment regimens for disorders associated with abnormal tooth eruption.