Lala R. Chaudhary

Erk is essential for growth, differentiation, integrin expression,and cell function in human osteoblastic cells

Extracellular signal-regulated kinases (Erks), members of the mitogen-activated protein kinase superfamily, play an important role in cell proliferation and differentiation. In this study we employed a dominant negative approach to determine the role of Erks in the regulation of human osteoblastic cell function. Human osteoblastic cells were transduced with a pseudotyped retrovirus encoding either a mutated Erk1 protein with a dominant negative action against both Erk1 and Erk2 (Erk1DN cells) or the LacZ protein (LacZ cells) as a control. Both basal and growth factor-stimulated MAPK activity and cell proliferation were inhibited in Erk1DN cells. Expression of Erk1DN protein suppressed both osteoblast differentiation and matrix mineralization by decreasing alkaline phosphatase activity and the deposition of bone matrix proteins. Cell adhesion to collagen, osteopontin, and vitronectin was decreased in Erk1DN cells as compared with LacZ cells. Cell spreading and migration on these matrices were also inhibited. In Erk1DN cells, expression of αβ1, αvβ3, and αvβ5 integrins on the surface was decreased. Metabolic labeling indicated that the synthesis of these integrins was inhibited in Erk1DN cells. These data suggest that Erks are not only essential for the growth and differentiation of osteoblasts but also are important for osteoblast adhesion, spreading, migration, and integrin expression.

The Mechanism of Phosphorus as a Cardiovascular Risk Factor in CKD

Hyperphosphatemia and vascular calcification have emerged as cardiovascular risk factors among those with chronic kidney disease. This study examined the mechanism by which phosphorous stimulates vascular calcification, as well as how controlling hyperphosphatemia affects established calcification. In primary cultures of vascular smooth muscle cells derived from atherosclerotic human aortas, activation of osteoblastic events, including increased expression of bone morphogenetic protein 2 (BMP-2) and the transcription factor RUNX2, which normally play roles in skeletal morphogenesis, was observed. These changes, however, did not lead to matrix mineralization until the phosphorus concentration of the media was increased; phosphorus stimulated expression of osterix, a second critical osteoblast transcription factor. Knockdown of osterix with small interference RNA (siRNA) or antagonism of BMP-2 with noggin prevented matrix mineralization in vitro. Similarly, vascular BMP-2 and RUNX2 were upregulated in atherosclerotic mice, but significant mineralization occurred only after the induction of renal dysfunction, which led to hyperphosphatemia and increased aortic expression of osterix. Administration of oral phosphate binders or intraperitoneal BMP-7 decreased expression of osterix and aortic mineralization. It is concluded that, in chronic kidney disease, hyperphosphatemia stimulates an osteoblastic transcriptional program in the vasculature, which is mediated by osterix activation in cells of the vascular tunica media and neointima.

Vitamin D Receptor Activators Can Protect against Vascular Calcification

An apparent conflict exists between observational studies that suggest that vitamin D receptor (VDR) activators provide a survival advantage for patients with ESRD and other studies that suggest that they cause vascular calcification. In an effort to explain this discrepancy, we studied the effects of the VDR activators calcitriol and paricalcitol on aortic calcification in a mouse model of chronic kidney disease (CKD)-stimulated atherosclerotic cardiovascular mineralization. At dosages sufficient to correct secondary hyperparathyroidism, calcitriol and paricalcitol were protective against aortic calcification, but higher dosages stimulated aortic calcification. At protective dosages, the VDR activators reduced osteoblastic gene expression in the aorta, which is normally increased in CKD, perhaps explaining this inhibition of aortic calcification. Interpreting the results obtained using this model, however, is complicated by the adynamic bone disorder; both calcitriol and paricalcitol stimulated osteoblast surfaces and rates of bone formation. Therefore, the skeletal actions of the VDR activators may have contributed to their protection against aortic calcification. We conclude that low, clinically relevant dosages of calcitriol and paricalcitol may protect against CKD-stimulated vascular calcification.

Inhibition of cell survival signal protein kinase B/Akt by curcumin in human prostate cancer cells

Although curcumin has been shown to inhibit prostate tumor growth in animal models, its mechanism of action is not clear. To better understand the anti‐cancer effects of curcumin, we investigated the effects of curcumin on cell survival factor Akt in human prostate cancer cell lines, LNCaP, PC‐3, and DU‐145. Our results demonstrated differential activation of Akt. Akt was constitutively activated in LNCaP and PC‐3 cells. Curcumin inhibited completely Akt activation in both LNCaP and PC‐3 cells. The presence of 10% serum decreased the inhibitory effect of curcumin in PC‐3 cells whereas complete inhibition was observed in 0.5% serum. Very little or no activation of Akt was observed in serum starved DU‐145 cells (0.5% serum). The presence of 10% serum activated Akt in DU‐145 cells and was not inhibited by curcumin. Results suggest that one of the mechanisms of curcumin inhibition of prostate cancer may be via inhibition of Akt. To our knowledge this is the first report on the curcumin inhibition of Akt activation in LNCaP and PC‐3 but not in DU‐145 cells. J. Cell. Biochem. 89: 1–5, 2003.

The cell survival signal Akt is differentially activated by PDGF‐BB, EGF, and FGF‐2 in osteoblastic cells

Stimulation of osteoblast survival signals may be an important mechanism of regulating bone anabolism. Protein kinase B (PKB/Akt), a serine‐threonine protein kinase, is a critical regulator of normal cell growth, cell cycle progression, and cell survival. In this study we have investigated the signaling pathways activated by growth factors PDGF‐BB, EGF, and FGF‐2 and determined whether PDGF‐BB, EGF, and FGF‐2 activated Akt in human or mouse osteoblastic cells. The results demonstrated that both ERK1 and ERK2 were activated by FGF‐2 and PDGF‐BB. Activation of ERK1 and ERK2 by PDGF‐BB and FGF‐2 was inhibited by PD 098059 (100 μM), a specific inhibitor of MEK. Wortmannin (500 nM), a specific inhibitor of phosphatidylinositol 3‐kinase ( PI 3‐K), inhibited the activation of ERK1 and ERK2 by PDGF‐BB but not by FGF‐2 suggesting that PI 3‐K mediated the activation of ERK MAPK pathway by PDGF‐BB but not by FGF‐2. Rapamycin, an inhibitor of p70 S6 protein kinase and a downstream target of ERK1/2 and PI 3‐K, did not affect the activation of ERK1 and ERK2 by the growth factors. Furthermore, our results demonstrated that Akt, a downstream target of PI 3‐K, was activated by PDGF‐BB but not by FGF‐2. Akt activation by PDGF‐BB was inhibited by PI 3‐kinase inhibitor LY294002. Rapamycin had no effect on Akt activation. Epidermal growth factor (EGF) also activated Akt in osteoblastic cells which was inhibited by LY294002 but not by rapamycin. Taken together, our data for the first time revealed that the activation of ERK1/2 by PDGF‐BB is mediated by PI 3‐K, and secondly, Akt is activated by PDGF‐BB and EGF but not by FGF‐2 in human and mouse osteoblastic cells. These results are of critical importance in understanding the role of these growth factors in apoptosis and cell survival. PDGF‐BB and EGF but not FGF‐2 may stimulate osteoblast cell survival. J. Cell. Biochem. 81:304–311, 2001.

Regulation of Interleukin-8 Gene Expression by Interleukin-1β, Osteotropic Hormones, and Protein Kinase Inhibitors in Normal Human Bone Marrow Stromal Cells

Interleukin-8 (IL-8), a potent neutrophil chemotactic peptide that elicits pleiotropic biological effects is secreted in large amounts by normal human osteoblastic and bone marrow osteoprogenitor stromal (HBMS) cells in response to IL-1β and tumor necrosis factor-α. In the present study we investigated the regulation of IL-8 gene expression by IL-1β, osteotropic hormones, and protein kinase inhibitors in primary cultures of HBMS cells. The treatment of HBMS cells with IL-1β increased the steady-state levels of IL-8 mRNA in a dose- and time-dependent fashion and was detectable within 1 h, reached maximal by 4 h, and remained elevated at 24 h, whereas parathyroid hormone (10−7 and 10−8 M) had no effect on IL-8 mRNA. Both synthetic and natural glucocorticoids dexamethasone (10−7-10−10 M) and hydrocortisone (10−6-10−8 M) inhibited IL-1β-stimulated IL-8 mRNA expression. The suppressive effect of dexamethasone on IL-1β-induced IL-8 mRNA was not observed in the presence of cycloheximide (5 μg/ml), indicating that the dexamethasone-mediated repression of IL-8 gene expression also depends on new protein synthesis. Experiments with actinomycin D demonstrated that IL-8 mRNA is long-lived and that glucocorticoids down-regulate IL-8 gene expression mainly by decreasing the mRNA stability in normal HBMS cells. Furthermore, as determined by nuclear run-on analysis, IL-1β increased the rate of transcription of IL-8 gene and dexamethasone did not affect the IL-1β-induced transcription of IL-8. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine, HCl (50 μM) and staurosporine (1 μM), potent inhibitors of protein kinase C, and genistein (100 μM), a specific protein tyrosine kinase inhibitor blocked IL-1β-induced IL-8 gene expression. Because curcumin (20 μM), an inhibitor of c-jun/AP-1 and protein kinases, also blocked IL-1β-stimulated IL-8 gene expression implicating c-JUN/AP-1 and protein phosphorylation in the induction of IL-8 gene expression by IL-1β, we conclude that the regulation of IL-8 mRNA by IL-1β is mediated via protein kinase-dependent signal transduction pathways. Our accumulated results have demonstrated that glucocorticoid suppression of IL-1β-induced IL-8 mRNA occurs at the levels of post-transcription (mRNA stability) and protein synthesis.

Extracellular-signal regulated kinase signaling pathway mediates downregulation of type I procollagen gene expression by FGF-2, PDGF-BB, and okadaic acid in osteoblastic cells.

Although basic fibroblast growth factor (FGF‐2) had been shown to inhibit type I collagen gene expression in osteoblast, its inhibitory mechanism is unknown. In the present study, we investigated the underlying mechanisms by which growth factors downregulate type I collagen gene expression. Treatment of mouse osteoblastic MC3T3‐E1 cells with okadaic acid (40 ng/ml), an inhibitor of phosphoserine/threonine‐specific protein phosphatase and activator of ERK1/2, for 24 h and 48 h completely inhibited steady‐state mRNA levels of type I collagen. FGF‐2 (30 ng/ml), platelet‐derived growth factor‐BB (PDGF‐BB), 30 ng/ml, and serum, which activate ERK mitogen‐activated protein kinase (MAPK) pathway also inhibited collagen type I gene expression, suggesting that the activation of ERK pathway mediates inhibition of type I collagen mRNA. This observation was further confirmed by experiments using inhibitors of the ERK pathway (i.e., PD and U0126), which increased type I collagen mRNA in MC3T3‐E1 cells, indicating that the inhibition of ERK pathway upregulates type I collagen gene expression. Low serum (0.3%) markedly increased type I collagen mRNA. MEK inhibitor PD inhibited c‐fos induction by FGF‐2 and PDGF‐BB, suggesting that c‐fos is the downstream target of ERK pathway. Our data have clearly demonstrated for the first time that the ERK MAPK pathway play an important role in the regulation of type I collagen gene expression in osteoblastic cells. Results also showed that one of the mechanisms by which FGF‐2 and PDGF‐BB downregulate type I collagen gene expression in the osteoblast is through the activation of ERK signaling pathway. J. Cell. Biochem. 76:354–359, 2000.

Proprotein convertases regulate activity of prostate epithelial cell differentiation markers and are modulated in human prostate cancer cells

Prostate derived factor (PDF) is a member of transforming growth factor‐β (TGF‐β) superfamily proteins involved in differentiation of the prostate epithelium. Proprotein convertases (PCs) such as furin are thought to mediate the processing of TGF‐β superfamily. In the present study, we demonstrated for the first time that human prostate cancer cell lines differentially synthesize and secret prostate derived factor (PDF), and that PDF secreted by LNCaP is processed by PCs. Exposure of LNCaP cells to the decanoyl‐Arg‐Val‐Lys‐Arg‐chloromethylketone (CMK), a synthetic furin‐like protease inhibitor, inhibited PDF processing and resulted in the loss of luminal cell phenotype and induction of basal cell phenotype in LNCaP cells as demonstrated by alternations in the expression of cytokeratins 8, 14, 18, and 19, markers of prostate epithelial cell differentiation. These results suggest that proprotein convertases may be involved in the regulation of prostate epithelial cell differentiation, and may be an important target of prostate cancer therapy. J. Cell. Biochem. 88: 394–399, 2003.

Induction of early growth response-1 gene by interleukin-1β and tumor necrosis factor-α in normal human bone marrow stromal and osteoblastic cells: regulation by a protein kinase C inhibitor

The early growth response-1 (Egr-1) gene has been identified as a nuclear transcriptional factor and implicated in the regulation of growth and differentiation of osteoblastic cells. In the present study, we investigated whether Egr-1 mRNA is expressed and induced by interleukin-1 β (IL-β) and tumor necrosis factor-α (TNF-α) in normal human bond marrow stromal (HBMS) and osteoblastic (HOB) cells. Results demonstrate a very low basal expression of Egr-1 mRNA which is induced by IL-1 β and TNF-α in a time- and dose-dependent manner. Egr-1 mRNA induction was detectable within 15 min, reached maximal by 60 min and thereafter declined to basal levels by 120 min. Induction of Egr-1 mRNA by IL-1β and TNF-α was completely inhibited by H-7 suggesting the mediation of protein kinase C. The induction by IL-1 β and TNF-α of Egr-1 mRNA was independent of de novo protein synthesis since this induction was also observed in the presence of protein synthesis inhibitor cycloheximide. Fetal bovine serum and cycloheximide also independently induced the Egr-1 mRNA. Actinomycin D experiments demonstrated that Egr-1 mRNA is degraded very rapidly with a half-life of 30 min. Our results demonstrate the expression of Egr-1 gene and its induction by IL-1β, and TNF-α in normal human bone marrow stromal (osteoprogenitor) and osteoblastic cells in primary cultures. Data also reveal that the expression of Egr-1 gene is inhibited by protein kinase C inhibitor H-7 suggesting that the activation of protein kinase C or other protein kinases resulting in the phosphorylation of specific transcription factor(s) is the first immediate early step in the induction of immediate-early Egr-1 gene by IL-1 β and TNF-α. Results also suggest that Egr-1 is an important mediator of IL-1 β and TNF-α action in normal human osteoblastic cells.

Activation of c‐Jun NH2‐terminal kinases by interleukin‐1β in normal human osteoblastic and rat UMR‐106 cells

We recently demonstrated the activation of extracellular signal‐ regulated protein kinase 1 and 2 (ERK1 and ERK2) by IGF‐1, FGF‐2, and PDGF‐BB in normal human osteoblastic (HOB) cells as well as in rat and mouse osteoblastic cells. In this report, we have examined whether c‐Jun NH2‐Terminal Kinase (JNK) pathway is activated by growth factors and interleukin‐1β (IL‐1β) in normal HOB and rat UMR‐106 cells using immune‐complex kinase assay and anti‐active JNK antibody, which recognizes activated forms of both JNK1 and JNK2. Results have demonstrated the presence of JNK1 and JNK2 proteins in normal HOB and UMR‐106 cells. Both JNK1 and JNK2 were activated by IL‐1β. IL‐1β preferentially activated JNK pathway in a dose‐ and time‐dependent manner and had little effect on ERK pathway. On the other hand, FGF‐2 did not activate JNK but most strongly activated ERK pathway. The activation of JNK was maximal at 20 min whereas maximal activation of ERK1 and ERK2 was observed within 10 min. Results have clearly demonstrated that IL‐1β preferentially activates JNK pathway whereas FGF‐2 activates ERK pathway in normal human and rat UMR‐106 osteoblastic cells. J. Cell. Biochem. 69:87–93, 1998.