Biomedical subjects

Majd Zayzafoon

Publications and source records attributed to Majd Zayzafoon.

11 recordsLinked to original sources

PubMed2006

Beta2-microglobulin promotes the growth of human renal cell carcinoma through the activation of the protein kinase A, cyclic AMP-responsive element-binding protein, and vascular endothelial growth factor axis.

PURPOSE: Beta(2)-microglobulin (beta2M), a soluble protein secreted by cancer and host inflammatory cells, has various biological functions, including antigen presentation. Because aberrant expression of beta2M has been reported in human renal cell carcinoma, we investigated the effects of beta2M overexpression on cancer cell growth and analyzed its molecular signaling pathway. EXPERIMENTAL DESIGN: We established clonal cell lines that overexpressed beta2M in human renal cell carcinoma (SN12C) cells and then examined cell growth in vitro and in vivo and studied the beta2M-mediated downstream cell signaling pathway. RESULTS: Our results showed that beta2M expression positively correlates with (a) in vitro growth on plastic dishes and as Matrigel colonies, (b) cell invasion and migration in Boyden chambers, and (c) vascular endothelial growth factor (VEGF) expression and secretion by cells. We found, in addition, that beta2M mediates its action through increased phosphorylation of cyclic AMP-responsive element-binding protein (CREB) via the protein kinase A-CREB axis, resulting in increased VEGF expression and secretion. In convergence with this signal axis, beta2M overexpression also activated both phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase pathways. Beta2M overexpression induced accelerated growth of SN12C in mouse subcutis and bone. Interrupting the beta2M signaling pathway using small interfering RNA led to apoptosis with increased activation of caspase-3 and caspase-9 and cleaved poly(ADP-ribose) polymerase. CONCLUSIONS: Our results showed for the first time that the beta2M-protein kinase A-CREB-VEGF signaling axis plays a crucial role in support of renal cell carcinoma growth and progression and reveals a novel therapeutic target.

Animals↗

PubMed2006

beta2-microglobulin is a signaling and growth-promoting factor for human prostate cancer bone metastasis.

The protein factor beta2-microglobulin (beta2M), purified from the conditioned medium of human prostate cancer cell lines, stimulated growth and enhanced osteocalcin (OC) and bone sialoprotein (BSP) gene expression in human prostate cancer cells by activating a cyclic AMP (cAMP)-dependent protein kinase A signaling pathway. When beta2M was overexpressed in prostate cancer cells, it induced explosive tumor growth in mouse bone through increased phosphorylated cAMP-responsive element binding protein (CREB) and activated CREB target gene expression, including OC, BSP, cyclin A, cyclin D1, and vascular endothelial growth factor. Interrupting the beta2M downstream signaling pathway by injection of the beta2M small interfering RNA liposome complex produced an effective regression of previously established prostate tumors in mouse bone through increased apoptosis as shown by immunohistochemistry and activation of caspase-9, caspase-3, and cleavage of poly(ADP-ribose) polymerase. These results suggest that beta2M signaling is an attractive new therapeutic target for the treatment of lethal prostate cancer bone metastasis.

Animals↗

PubMed2006

Calcium/calmodulin signaling controls osteoblast growth and differentiation.

Ca2+ is a ubiquitous intracellular messenger responsible for controlling numerous cellular processes including fertilization, mitosis, neuronal transmission, contraction and relaxation of muscles, gene transcription, and cell death. At rest, the cytoplasmic Ca2+ concentration [Ca2+]i is approximately 100 nM, but this level rises to 500-1,000 nM upon activation. In osteoblasts, the elevation of [Ca2+]i is a result of an increase in the release of Ca2+ from endoplasmic reticulum and/or extracellular Ca2+ influx through voltage gated Ca2+ channels. Many of the cellular effects of Ca2+ are mediated by the Ca2+ binding protein, calmodulin (CaM). Upon binding up to four calcium ions, CaM undergoes a conformational change, which enables it to bind to specific proteins eliciting a specific response. Calmodulin kinase II (CaMKII) is a major target of the Ca(2+)/CaM second messenger system. Once bound to Ca(2+)/CaM, the multimeric CaMKII is released from its autoinhibitory status and maximally activated, which then leads to an intraholoenzyme autophosphorylation reaction. Calcineurin (Cn) is another major target protein that is activated by Ca(2+)/CaM. Cn is a serine-threonine phosphatase that consists of a heterodimeric protein complex composed of a catalytic subunit (CnA) and a regulatory subunit (CnB). Upon activation, Cn directly binds to, and dephosphorylates nuclear factor of activated T cells (NFAT) transcription factors within the cytoplasm allowing them to translocate to the nucleus and participate in the regulation of gene expression. This review will examine the potential mechanisms by which calcium, CaM, CaMKII, and Cn/NFAT control osteoblast proliferation and differentiation.

Animals↗

PubMed2006

NFATc1: a novel anabolic therapeutic target for osteoporosis.

Bone loss and osteoporosis are major public health problems in the elderly. With increasing life expectancy in the United States, the number of people that will develop age-related bone loss and osteoporosis is expected to rise to over 61 million by 2020. Osteoblast differentiation is a crucial aspect of bone formation and remodeling, a process severely compromised in osteoporosis. Almost all the FDA-approved treatments for building healthier bones, excluding parathyroid hormone (PTH), do not address the decrease in osteoblast differentiation seen in osteoporosis and rather are designed to target osteoclasts and bone resorption. The purpose of this study is to examine the effects of NFAT inhibition on osteoblast differentiation and to elucidate the mechanism of its action. Here we demonstrate that the inhibition of calcineurin (Cn) by using cyclosporine A (CsA) increases osteoblast differentiation, both in vivo and in vitro. Furthermore, the specific inhibition of NFATc1 by siRNA increased Fra-2 expression in osteoblasts. Taken together, our results point the way to a novel mechanism to aid in the development of anabolic treatment for osteoporosis.

Animals↗

PubMed2005

RhoA and cytoskeletal disruption mediate reduced osteoblastogenesis and enhanced adipogenesis of human mesenchymal stem cells in modeled microgravity.

UNLABELLED: Spaceflight, aging, and disuse lead to reduced BMD. This study shows that overexpression of constitutively active RhoA restores actin cytoskeletal arrangement, enhances the osteoblastic phenotype, and suppresses the adipocytic phenotype of human mesenchymal stem cells cultured in modeled microgravity. INTRODUCTION: Reduced BMD during spaceflight is partly caused by reduced bone formation. However, mechanisms responsible for this bone loss remain unclear. We have previously shown reduced osteoblastogenesis and enhanced adipogenesis of human mesenchymal stem cells (hMSCs) cultured in modeled microgravity (MMG). The small GTPase, RhoA, regulates actin stress fiber formation and has been implicated in the lineage commitment of hMSCs. We examined the effects of MMG on actin cytoskeletal organization and RhoA activity and the ability of constitutively active RhoA to reverse these effects. MATERIALS AND METHODS: hMSCs were seeded onto plastic microcarrier beads at a density of 10(6) and allowed to form aggregates in DMEM containing 10% FBS for 7 days. Aggregates were incubated in DMEM containing 2% FBS for 6 h with or without an adenoviral vector containing constitutively active RhoA at a multiplicity of infection (moi) of 500 and allowed to recover in 10% FBS for 24 h. Cells were transferred to the rotary cell culture system to model microgravity or to be maintained at normal gravity for 7 days in DMEM, 10% FBS, 10 nM dexamethasone, 10 mM beta-glycerol phosphate, and 50 muM ascorbic acid 2-phosphate. RESULTS: F-actin stress fibers are disrupted in hMSCs within 3 h of initiation of MMG and are completely absent by 7 days, whereas monomeric G-actin is increased. Because of the association of G-actin with lipid droplets in fat cells, the observed 310% increase in intracellular lipid accumulation in hMSCs cultured in MMG was not unexpected. Consistent with these changes in cellular morphology, 7 days of MMG significantly reduces RhoA activity and subsequent phosphorylation of cofilin by 88+/-2% and 77+/-9%, respectively. Importantly, introduction of an adenoviral construct expressing constitutively active RhoA reverses the elimination of stress fibers, significantly increases osteoblastic gene expression of type I collagen, alkaline phosphatase, and runt-related transcription factor 2, and suppresses adipocytic gene expression of leptin and glucose transporter 4 in hMSCs cultured in MMG. CONCLUSION: Suppression of RhoA activity during MMG represents a novel mechanism for reduced osteoblastogenesis and enhanced adipogenesis of hMSCs.

Adipogenesis↗

PubMed2005

Microgravity: the immune response and bone.

Exposure to microgravity during space flight affects almost all human physiological systems. The affected systems that are of key importance to human space exploration are the musculoskeletal, neurovestibular, and cardiovascular systems. However, alterations in the immune and endocrine functions have also been described. Bone loss has been shown to be site specific, predominantly in the weight-bearing regions of the legs and lumbar spine. This phenomenon has been attributed to a reduction in bone formation resulting from a decrease in osteoblastic function and an increase in osteoclastic resorption. In order to examine the effects of microgravity on cellular function here on earth, several ground-based studies have been performed using different systems to model microgravity. Our studies have shown that modeled microgravity (MMG) inhibits the osteoblastic differentiation of human mesenchymal stem cells (hMSCs) while increasing their adipogenic differentiation. Here, we discuss the potential molecular mechanisms that could be altered in microgravity. In particular, we examine the role of RhoA kinase in maintaining the formation of actin stress fibers and the expression of nitric oxide synthase under MMG conditions. These proposed mechanisms, although only examined in hMSCs, could be part of a global response to microgravity that ultimately alters human physiology.

Animals↗

PubMed2004

Calmodulin and calmodulin-dependent kinase IIalpha regulate osteoblast differentiation by controlling c-fos expression.

Ca(2+)/calmodulin-dependent protein kinase IIalpha (alpha-CaMKII) was once thought to be exclusively expressed in neuronal tissue, but it is becoming increasingly evident that CaMKII is also expressed in various extraneural cells. CaMKII plays a critical role in regulating various signaling pathways leading to modulation of several aspects of cellular functions, including proliferation, differentiation, cytoskeletal structure, and gene expression. The purpose of this study was to examine the expression of CaMKII in osteoblast-like cells (MC4) and to elucidate its role in osteoblast differentiation. We demonstrated that CaMKII, specifically the alpha isoform, is expressed in osteoblasts both in vitro and in vivo. Inhibition of CaMKII by the calmodulin antagonist trifluoperazine or the CaMKII antagonist KN93 reduces alkaline phosphatase activity and mineralization, as well as causes 85 and 56% decreases in alkaline phosphatase and osteocalcin gene expression, respectively. CaM and CaMKII antagonists, using the newborn mouse calvaria in vivo model, cause a 50% decrease in osteoblast number (N.Ob-BS) and a 32% decrease in mineralization (BV/TV). Pharmacologic and genetic inhibition of alpha-CaMKII by using trifluoperazine, KN93, and alpha-CaMKII small interfering RNA decreases the phosphorylation of ERK and of cAMP-response element-binding protein, leading to a significant decrease in the transactivation of serum response element and cAMP-response element. Inhibition of alpha-CaMKII decreases the expression of c-fos, AP-1 transactivation, and AP-1 DNA binding activity. Our findings demonstrated that alpha-CaMKII is expressed in osteoblasts and is involved in c-fos expression via regulation of serum response element and cAMP-response element. Inhibition of alpha-CaMKII results in a decrease in c-fos expression and AP-1 activation, leading to inhibition of osteoblast differentiation.

Animals↗

PubMed2004

RANKL regulates Fas expression and Fas-mediated apoptosis in osteoclasts.

UNLABELLED: Osteoclast apoptosis is an influential determinant of osteoclast bone-resorbing activity. RANKL, a critical factor for osteoclastogenesis, is also important in osteoclast survival. However, the mechanisms by which RANKL prevents osteoclast apoptosis remain largely unknown. INTRODUCTION: Fas, a death receptor, mediates apoptosis in multiple types of cells including osteoclasts. Here we report that RANKL acts as a survival factor in osteoclasts by downregulating Fas-mediated apoptosis and Fas expression in mature osteoclasts. MATERIALS AND METHODS: RAW264.7 and mouse bone marrow macrophage/monocyte progenitors and progenitor-derived osteoclasts, in the presence of various concentrations of RANKL, were used in this study. Western blotting, semiquantitative RT-PCR, flow cytometry, nuclear staining, and a fluorescent caspase-3 activity assay were used to assess the effect of RANKL on Fas expression and Fas-mediated apoptosis. The involvement of NF-kappaB in the regulation of Fas by RANKL was analyzed by luciferase assay and EMSA. RESULTS: Mature osteoclasts generated in the presence of a high concentration of RANKL (3.33 nM) failed to respond to Fas-induced apoptosis. The lack of responsiveness in mature osteoclasts is caused by the low level of Fas expression, as detected by both semiquantitative PCR and Western blotting. Fas protein and mRNA expression are inhibited by RANKL in concentration-dependent manners. The downregulation of Fas expression by RANKL is not because of modulation of the stability of Fas protein or mRNA. The regulation of Fas expression by RANKL is biphasic. During the early stage of osteoclastogenesis (1 day) when Fas is expressed at a very low level, RANKL upregulates Fas promoter activity by 2.4 +/- 0.1-fold in a concentration-dependent manner and increases Fas mRNA and protein. This event correlates with regulation of the binding activity of NF-kappaB to the Fas promoter by RANKL, as detected by EMSA. In osteoclast precursors, the induction of Fas promoter activity by RANKL was dramatically reduced when NF-kappaB binding sites on the Fas promoter were mutated. CONCLUSION: RANKL upregulates Fas expression in osteoclast progenitors through NF-kappaB, making osteoclasts targets of Fas-stimulated apoptosis. In differentiated mature osteoclasts, RANKL reduces the levels of Fas expression and Fas-mediated apoptosis, acting as a survival factor.

Animals↗

PubMed2004

Modeled microgravity inhibits osteogenic differentiation of human mesenchymal stem cells and increases adipogenesis.

Space flight-induced bone loss has been attributed to a decrease in osteoblast function, without a significant change in bone resorption. To determine the effect of microgravity (MG) on bone, we used the Rotary Cell Culture System [developed by the National Aeronautics and Space Administration (NASA)] to model MG. Cultured mouse calvariae demonstrated a 3-fold decrease in alkaline phosphatase (ALP) activity and failed to mineralize after 7 d of MG. ALP and osteocalcin gene expression were also decreased. To determine the effects of MG on osteoblastogenesis, we cultured human mesenchymal stem cells (hMSC) on plastic microcarriers, and osteogenic differentiation was induced immediately before the initiation of modeled MG. A marked suppression of hMSC differentiation into osteoblasts was observed because the cells failed to express ALP, collagen 1, and osteonectin. The expression of runt-related transcription factor 2 was also inhibited. Interestingly, we found that peroxisome proliferator-activated receptor gamma (PPARgamma2), which is known to be important for adipocyte differentiation, adipsin, leptin, and glucose transporter-4 are highly expressed in response to MG. These changes were not corrected after 35 d of readaptation to normal gravity. In addition, MG decreased ERK- and increased p38-phosphorylation. These pathways are known to regulate the activity of runt-related transcription factor 2 and PPARgamma2, respectively. Taken together, our findings indicate that modeled MG inhibits the osteoblastic differentiation of hMSC and induces the development of an adipocytic lineage phenotype. This work will increase understanding and aid in the prevention of bone loss, not only in MG but also potentially in age-and disuse-related osteoporosis.

Adipocytes↗

PubMed2003

Notch signaling and ERK activation are important for the osteomimetic properties of prostate cancer bone metastatic cell lines.

Prostate cancer bone metastases are characterized by their ability to induce osteoblastic lesions and local bone formation. It has been suggested that bone metastatic prostate cancer cells are osteomimetic and capable of expressing genes and proteins typically expressed by osteoblasts. The ability of preosteoblasts to differentiate and express osteoblastic genes depends on several pathways, including Notch and MAPK. Here we show that notch1 expression is increased 4-5 times in C4-2B and MDA PCa 2b cells (osteoblastic skeletal prostate metastatic cancer cell lines) when compared with nonskeletal metastatic cell lines (LNCaP and DU145). Notch1 ligand, dll1, is expressed only in C4-2B cells. Immunohistochemical studies demonstrate that Notch1 is present in both human clinical samples from prostate cancer bone metastases and the C4-2B cell line. To determine whether prostate cancer bone metastases respond to osteogenic induction similar to osteoblasts, C4-2B cells were cultured in osteogenic medium that promotes mineralization. C4-2B cells mineralize and express HES-1 (a downstream target of Notch), an effect that is completely inhibited by L-685,458, a Notch activity inhibitor. Furthermore, osteogenic induction increases ERK activation, runx2 expression, and nuclear localization, independent of Notch signaling. Finally, we show that Notch and ERK activation are essential for Runx2 DNA binding activity and osteocalcin gene expression in C4-2B cells in response to osteogenic induction. These studies demonstrate that prostate cancer bone metastatic cell lines acquire osteoblastic properties through independent activation of ERK and Notch signaling; presumably, both pathways are activated in the bone microenvironment.

Basic Helix-Loop-Helix Proteins↗

PubMed2002

P38 and activating transcription factor-2 involvement in osteoblast osmotic response to elevated extracellular glucose.

Poorly controlled or untreated type I diabetes mellitus is characterized by hyperglycemia and is associated with decreased bone mass and osteoporosis. We have demonstrated that osteoblasts are sensitive to hyperglycemia-associated osmotic stress and respond to elevated extracellular glucose or mannitol by increasing c-jun and collagen I expression. To determine whether MAPKs are involved in this response, MC3T3-E1 osteoblasts were treated with 16.5 mm glucose, mannitol, or contrast dye for 1 h. Immunoblotting of phosphorylated p38 demonstrated activation of p38 MAPK by hyperosmotic stress in vitro and in vivo. Activation peaked at 20 min, remained detectable after 24 h, and was protein kinase C-independent. Activating transcription factor-2 (ATF-2) activation followed the same pattern as phospho-p38. Transactivation of cAMP response element (CRE)- and c-jun promoter (containing a CRE-like element)-reporter constructs increased following hyperosmotic treatment. SB 203580 (a p38 MAPK inhibitor) blocked ATF-2 phosphorylation, CRE transactivation, and c-jun promoter activation. Hyperosmotic activation of collagen I promoter activity was also inhibited by SB 203580, consistent with the involvement of c-jun in collagen I up-regulation. Therefore, we propose that hyperglycemia-induced increases in p38 MAPK activity and ATF-2 phosphorylation contribute to CRE activation and modulation of c-jun and collagen I expression in osteoblasts.

3T3 Cells↗