Majd Zayzafoon

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 Ca2+/CaM second messenger system. Once bound to Ca2+/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 Ca2+/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. J. Cell. Biochem.

RhoA and Cytoskeletal Disruption Mediate Reduced Osteoblastogenesis and Enhanced Adipogenesis of Human Mesenchymal Stem Cells in Modeled Microgravity

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.

T lymphocytes amplify the anabolic activity of parathyroid hormone through Wnt10b signaling.

Intermittent administration of parathyroid hormone (iPTH) is used to treat osteoporosis because it improves bone architecture and strength, but the underlying cellular and molecular mechanisms are unclear. Here, we show that iPTH increases the production of Wnt10b by bone marrow CD8+ T cells and induces these lymphocytes to activate canonical Wnt signaling in preosteoblasts. Accordingly, in responses to iPTH, T cell null mice display diminished Wnt signaling in preosteoblasts and blunted osteoblastic commitment, proliferation, differentiation, and life span, which result in decreased trabecular bone anabolism and no increase in strength. Demonstrating the specific role of lymphocytic Wnt10b, iPTH has no anabolic activity in mice lacking T-cell-produced Wnt10b. Therefore, T-cell-mediated activation of Wnt signaling in osteoblastic cells plays a key permissive role in the mechanism by which iPTH increases bone strength, suggesting that T cell osteoblast crosstalk pathways may provide pharmacological targets for bone anabolism.

Receptor activator of NF-κB Ligand (RANKL) expression is associated with epithelial to mesenchymal transition in human prostate cancer cells

Epithelial-mesenchymal transition (EMT) in cancer describes the phenotypic and behavioral changes of cancer cells from indolent to virulent forms with increased migratory, invasive and metastatic potential. EMT can be induced by soluble proteins like transforming growth factor β1 (TGFβ1) and transcription factors including Snail and Slug. We utilized the ARCaPE/ARCaPM prostate cancer progression model and LNCaP clones stably overexpressing Snail to identify novel markers associated with EMT. Compared to ARCaPE cells, the highly tumorigenic mesenchymal ARCaPM and ARCaPM1 variant cells displayed a higher incidence of bone metastasis after intracardiac administration in SCID mice. ARCaPM and ARCaPM1 expressed mesenchymal stromal markers of vimentin and N-cadherin in addition to elevated levels of Receptor Activator of NF-κB Ligand (RANKL). We observed that both epidermal growth factor (EGF) plus TGFβ1 treatment and Snail overexpression induced EMT in ARCaPE and LNCaP cells, and EMT was associated with increased expression of RANKL protein. Finally, we determined that the RANKL protein was functionally active, promoting osteoclastogenesis in vitro. Our results indicate that RANKL is a novel marker for EMT during prostate cancer progression. RANKL may function as a link between EMT, bone turnover, and prostate cancer skeletal metastasis.

Modeled microgravity disrupts collagen I/integrin signaling during osteoblastic differentiation of human mesenchymal stem cells

Spaceflight leads to reduced bone mineral density in weight bearing bones that is primarily attributed to a reduction in bone formation. We have previously demonstrated severely reduced osteoblastogenesis of human mesenchymal stem cells (hMSC) following 7 days culture in modeled microgravity (MMG). One potential mechanism for reduced osteoblastic differentiation is disruption of type I collagen (Col I)–integrin interactions and reduced integrin signaling. Integrins are heterodimeric transmembrane receptors that bind extracellular matrix (ECM) proteins and produce signals essential for proper cellular function, survival, and differentiation. Therefore, we investigated the effects of MMG on integrin expression and function in hMSC. We demonstrate that 7 days of culture in MMG leads to reduced expression of the ECM protein, Col I. Conversely, MMG consistently increases Col I‐specific α2 and β1 integrin protein expression. Despite this increase in integrin subunit expression, autophosphorylation of adhesion‐dependent kinases, focal adhesion kinase (FAK) and proline‐rich tyrosine kinase 2 (PYK2), is significantly reduced. Activation of Akt protein kinase (Akt) is unaffected by the reduction in FAK activation. However, reduced downstream signaling via the Ras‐mitogen activated protein kinase (MAPK) pathway is evidenced by a reduction in Ras and extracellular signal‐related protein kinase (ERK) activation. Taken together, our findings indicate that MMG decreases integrin/MAPK signaling, which likely contributes to the observed reduction in osteoblastogenesis.

Ovariectomy disregulates osteoblast and osteoclast formation through the T-cell receptor CD40 ligand

The bone loss induced by ovariectomy (ovx) has been linked to increased production of osteoclastogenic cytokines by bone marrow cells, including T cells and stromal cells (SCs). It is presently unknown whether regulatory interactions between these lineages contribute to the effects of ovx in bone, however. Here, we show that the T-cell costimulatory molecule CD40 ligand (CD40L) is required for ovx to expand SCs; promote osteoblast proliferation and differentiation; regulate the SC production of the osteoclastogenic factors macrophage colony-stimulating factor, receptor activator of nuclear factor-κB ligand, and osteoprotegerin; and up-regulate osteoclast formation. CD40L is also required for ovx to activate T cells and stimulate their production of TNF. Accordingly, ovx fails to promote bone loss and increase bone resorption in mice depleted of T cells or lacking CD40L. Therefore, cross-talk between T cells and SCs mediated by CD40L plays a pivotal role in the disregulation of osteoblastogenesis and osteoclastogenesis induced by ovx.

Alterations in the immuno-skeletal interface drive bone destruction in HIV-1 transgenic rats.

Osteoporosis and bone fractures are increasingly recognized complications of HIV-1 infection. Although antiretroviral therapy itself has complex effects on bone turnover, it is now evident that the majority of HIV-infected individuals already exhibit reduced bone mineral density before therapy. The mechanisms responsible are likely multifactorial and have been difficult to delineate in humans. The HIV-1 transgenic rat recapitulates many key features of human AIDS. We now demonstrate that, like their human counterparts, HIV-1 transgenic rats undergo severe osteoclastic bone resorption, a consequence of an imbalance in the ratio of receptor activator of NF-κB ligand, the key osteoclastogenic cytokine, to that of its physiological decoy receptor osteoprotegerin. This imbalance stemmed from a switch in production of osteoprotegerin to that of receptor activator of NF-κB ligand by B cells, and was further compounded by a significantly elevated number of osteoclast precursors. With the advancing age of individuals living with HIV/AIDS, low bone mineral density associated with HIV infection is likely to collide with the pathophysiology of skeletal aging, leading to increased fracture risk. Understanding the mechanisms driving bone loss in HIV-infected individuals will be critical to developing effective therapeutic strategies.

Immunohistochemistry in the evaluation of neovascularization in tumor xenografts

Angiogenesis, or neovascularization, is known to play an important role in the neoplastic progression leading to metastasis. CD31 or Factor VIII-related antigen (F VIII RAg) immunohistochemistry is widely used in experimental studies for quantifying tumor neovascularization in immunocompromised animal models implanted with transformed human cell lines. Quantification, however, can be affected by variations in the methodology used to measure vascularization including antibody selection, antigen retrieval (AR) pretreatment, and evaluation techniques. To examine this further, we investigated the microvessel density (MVD) and the intensity of microvascular staining among five different human tumor xenografts and a mouse syngeneic tumor using anti-CD31 and F VIII RAg immunohistochemical staining. Different AR methods also were evaluated. Maximal retrieval of CD31 was achieved using 0.5 M Tris (pH 10) buffer, while maximum retrieval of F VIII RAg was achieved using 0.05% pepsin treatment of tissue sections. For each optimized retrieval condition, anti-CD31 highlighted small vessels better than F VIII RAg. Furthermore, the MVD of CD31 was significantly greater than that of F VIII RAg decorated vessels (p<0.001). The choice of antibody and AR method has a significant affect on immunohistochemical findings when studying angiogenesis. One also must use caution when comparing studies in the literature that use different techniques and reagents.

β2-microglobulin is a signaling and growth-promoting factor for human prostate cancer bone metastasis

The protein factor β2-microglobulin (β2M), 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 β2M 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 β2M downstream signaling pathway by injection of the β2M 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 β2M signaling is an attractive new therapeutic target for the treatment of lethal prostate cancer bone metastasis. (Cancer Res 2006; 66(18): 9108-16)

Honokiol, a natural plant product, inhibits the bone metastatic growth of human prostate cancer cells

Honokiol, a soluble nontoxic natural product derived from Magnolia spp., has been shown to induce apoptosis in malignant cells. The effect of honokiol and the combined therapy with docetaxel on prostate cancer (PCa) growth and bone metastasis was investigated in experimental models.