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 seven days culture in modeled microgravity. One potential mechanism for reduced osteoblastic differentiation is disruption of type I collagen-integrin interactions and reduced integrin signaling. Integrins are heterodimeric transmembrane receptors that bind extracellular matrix proteins and produce signals essential for proper cellular function, survival, and differentiation. Therefore, we investigated the effects of modeled microgravity on integrin expression and function in hMSC. We demonstrate that seven days of culture in modeled microgravity leads to reduced expression of the extracellular matrix protein, type I collagen (Col I). Conversely, modeled microgravity consistently increases Col I-specific alpha2 and beta1 integrin protein expression. Despite this increase in integrin sub-unit expression, autophosphorylation of adhesion-dependent kinases, focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2 (PYK2), is significantly reduced. Activation of Akt is unaffected by the reduction in FAK activation. However, reduced downstream signaling via the Ras-MAPK pathway is evidenced by a reduction in Ras and ERK activation. Taken together, our findings indicate that modeled microgravity decreases integrin/MAPK signaling, which likely contributes to the observed reduction in osteoblastogenesis

LIV-1 Promotes Prostate Cancer Epithelial-to-Mesenchymal Transition and Metastasis through HB-EGF Shedding and EGFR-Mediated ERK Signaling

LIV-1, a zinc transporter, is an effector molecule downstream from soluble growth factors. This protein has been shown to promote epithelial-to-mesenchymal transition (EMT) in human pancreatic, breast, and prostate cancer cells. Despite the implication of LIV-1 in cancer growth and metastasis, there has been no study to determine the role of LIV-1 in prostate cancer progression. Moreover, there was no clear delineation of the molecular mechanism underlying LIV-1 function in cancer cells. In the present communication, we found increased LIV-1 expression in benign, PIN, primary and bone metastatic human prostate cancer. We characterized the mechanism by which LIV-1 drives human prostate cancer EMT in an androgen-refractory prostate cancer cells (ARCaP) prostate cancer bone metastasis model. LIV-1, when overexpressed in ARCaPE (derivative cells of ARCaP with epithelial phenotype) cells, promoted EMT irreversibly. LIV-1 overexpressed ARCaPE cells had elevated levels of HB-EGF and matrix metalloproteinase (MMP) 2 and MMP 9 proteolytic enzyme activities, without affecting intracellular zinc concentration. The activation of MMPs resulted in the shedding of heparin binding-epidermal growth factor (HB-EGF) from ARCaPE cells that elicited constitutive epidermal growth factor receptor (EGFR) phosphorylation and its downstream extracellular signal regulated kinase (ERK) signaling. These results suggest that LIV-1 is involved in prostate cancer progression as an intracellular target of growth factor receptor signaling which promoted EMT and cancer metastasis. LIV-1 could be an attractive therapeutic target for the eradication of pre-existing human prostate cancer and bone and soft tissue metastases

Effect of HIP/ribosomal protein L29 deficiency on mineral properties of murine bones and teeth

Mice lacking HIP/RPL29, a component of the ribosomal machinery, display increased bone fragility. To understand the effect of sub-efficient protein synthetic rates on mineralized tissue quality, we performed dynamic and static histomorphometry and examined the mineral properties of both bones and teeth in HIP/RPL29 knock-out mice using Fourier transform infrared imaging (FTIRI). While loss of HIP/RPL29 consistently reduced total bone size, decreased mineral apposition rates were not significant, indicating that short stature is not primarily due to impaired osteoblast function. Interestingly, our microspectroscopic studies showed that a significant decrease in collagen crosslinking during maturation of HIP/RPL29-null bone precedes an overall enhancement in the relative extent of mineralization of both trabecular and cortical adult bones. This report provides strong genetic evidence that ribosomal insufficiency induces subtle organic matrix deficiencies which elevates calcification. Consistent with the HIP/RPL29-null bone phenotype, HIP/RPL29-deficient teeth also showed reduced geometric properties accompanied with relative increased mineral densities of both dentin and enamel. Increased mineralization associated with enhanced tissue fragility related to imperfection in organic phase microstructure evokes defects seen in matrix protein-related bone and tooth diseases. Thus, HIP/RPL29 mice constitute a new genetic model for studying the contribution of global protein synthesis in the establishment of organic and inorganic phases in mineral tissues

T Lymphocytes Amplify the Anabolic Activity of Parathyroid Hormone through Wnt10b Signaling

SummaryIntermittent 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

NFAT Signaling in Osteoblasts Regulates the Hematopoietic Niche in the Bone Microenvironment

Osteoblasts support hematopoietic cell development, including B lymphopoiesis. We have previously shown that the nuclear factor of activated T cells (NFAT) negatively regulates osteoblast differentiation and bone formation. Interestingly, in smooth muscle, NFAT has been shown to regulate the expression of vascular cellular adhesion molecule-1 (VCAM-1), a mediator of cell adhesion and signaling during leukocyte development. To examine whether NFAT signaling in osteoblasts regulates hematopoietic development in vivo, we generated a mouse model expressing dominant-negative NFAT driven by the 2.3 kb fragment of the collagen-αI promoter to disrupt NFAT activity in osteoblasts (dnNFATOB). Bone histomorphometry showed that dnNFATOB mice have significant increases in bone volume (44%) and mineral apposition rate (131%) and decreased trabecular thickness (18%). In the bone microenvironment, dnNFATOB mice displayed a significant increase (87%) in Lineage−cKit+Sca-1+ (LSK) cells and significant decreases in B220+CD19−IgM− pre-pro-B cells (41%) and B220+CD19+IgM+ immature B cells (40%). Concurrent with these findings, LSK cell differentiation into B220+ cells was inhibited when cocultured on differentiated primary osteoblasts harvested from dnNFATOB mice. Gene expression and protein levels of VCAM-1 in osteoblasts decreased in dnNFATOB mice compared to controls. These data suggest that osteoblast-specific NFAT activity mediates early B lymphopoiesis, possibly by regulating VCAM-1 expression on osteoblasts

The growth and aggressive behavior of human osteosarcoma is regulated by a CaMKII-controlled autocrine VEGF signaling mechanism.

Osteosarcoma (OS) is a hyperproliferative malignant tumor that requires a high vascular density to maintain its large volume. Vascular Endothelial Growth Factor (VEGF) plays a crucial role in angiogenesis and acts as a paracrine and autocrine agent affecting both endothelial and tumor cells. The alpha-Ca2+/Calmodulin kinase two (α-CaMKII) protein is an important regulator of OS growth. Here, we investigate the role of α-CaMKII-induced VEGF in the growth and tumorigenicity of OS. We show that the pharmacologic and genetic inhibition of α-CaMKII results in decreases in VEGF gene expression (50%) and protein secretion (55%), while α- CaMKII overexpression increases VEGF gene expression (250%) and protein secretion (1,200%). We show that aggressive OS cells (143B) express high levels of VEGF receptor 2 (VEGFR-2) and respond to exogenous VEGF (100nm) by increasing intracellular calcium (30%). This response is ameliorated by the VEGFR inhibitor CBO-P11, suggesting that secreted VEGF results in autocrine stimulated α-CaMKII activation. Furthermore, we show that VEGF and α-CaMKII inhibition decreases the transactivation of the HIF-1α and AP-1 reporter constructs. Additionally, chromatin immunoprecipitation assay shows significantly decreased binding of HIF-1α and AP-1 to their responsive elements in the VEGF promoter. These data suggest that α-CaMKII regulates VEGF transcription by controlling HIF-1α and AP-1 transcriptional activities. Finally, CBO-P11, KN-93 (CaMKII inhibitor) and combination therapy significantly reduced tumor burden in vivo. Our results suggest that VEGF-induced OS tumor growth is controlled by CaMKII and dual therapy by CaMKII and VEGF inhibitors could be a promising therapy against this devastating adolescent disease

VEGF expression is positively regulated by CaMKII in human OS.

<p>143B cells were transduced with lentiviruses expressing either scrambled (shCtrl) or α-CaMKII- targeting shRNAs (shCaMKIIα) or treated with the CaMKII inhibitor KN-93. HOS cells were transduced with retroviruses expressing either GFP (GFP-Ctrl) or CaMKIIα (GFP- CaMKIIα). <b>A.</b> Real-time PCR was performed using primers specific for VEGF or β-Actin. Values were obtained from three separate experiments each repeated in triplicate and represent the mean ±S.D. *p<0.01. <b>B.</b> α-CaMKII-inhibited 143B (shCaMKIIα or KN-93) and α-CaMKII overexpressing HOS (GFP-CaMKIIα) cells were seeded at 1 x 10⁵ cells per well of a 6-well plate. Twenty-four hours later, aliquots of supernatant were examined for human VEGF by ELISA. Values were obtained from three separate experiments each repeated in triplicate and represent the mean ±S.D. *p<0.01. <b>C.</b> α-CaMKII inhibited 143B (shCaMKIIα or KN-93) and α- CaMKII overexpressing HOS (GFP-CaMKIIα) cells were seeded at 1 x 10⁵ cells per well of a 6- well plate. Twenty-four hours later, aliquots of conditioned media were removed and added to 12-well plates seeded with 1 x 10⁵ HUVEC cells. Endothelial cell tube-like formation was measured at 12 h. Negative controls of non-conditioned culture media (DMEM + 10% FBS) and conditioned media with VEGF immunoprecipitated out (VEGF IP) were also included. Representative photomicrographs were taken at 50x magnification from 3 independent experiments, each repeated in triplicate. Scale bar = 50 μm <b>D.</b> Capillary tube-like length was quantified using the ImageJ software. Values were obtained from three separate experiments each repeated in triplicate and represent the mean ±S.D. *p<0.01.</p

CaMKII controls VEGF gene expression by regulating TRE and HRE.

<p><b>A.</b> Schematic illustration of the VEGF promoter showing the binding sites for the AP-1 and HIF-1α transcription factors <b>B.</b> Reporter activation assay. Cells were transfected with TRE and HRE luciferase constructs and treated with 10 μM KN-93 and/or 1 μM CBO-P11 for 24 hours and harvested 24 hours later for luciferase activity measurement. Data are expressed relative to total protein, and values represent the mean ± SD of 3 separate experiments each repeated in triplicate; *p≤ 0.01. <b>C.</b> Quantification of western blot analyses. 143B cells were treated with 10 μM KN-93 and/or 1 μM CBO-P11 for 24 hours. Immunoblots were developed using specific antibodies directed against p-c-Jun, c-Fos, HIF-1α, Lamin B1 or β-Actin. Band density was measured using imageJ software and normalized to β-Actin. <b>D.</b> ChIP assay. DNA-protein complexes were immunoprecipitated with antibodies against AP-1 (c-Fos), HIF-1α and normal rabbit IgG as a control. Immunoprecipitated DNA fragments were amplified by PCR using primers encompassing the TRE and HRE binding sites on the VEGF promoter. Three independent experiments were performed. <b>E.</b> Quantitative analyses of the endogenous VEGF Expression. Real-time PCR was performed using primers specific for VEGF or β-Actin in human OS cell lines treated with 10 μM KN-93 and/or 1μM CBO-P11 for 24 hours. Values were obtained from three separate experiments each replicated in triplicate and represent the mean ±S.D. *p<0.01.</p

VEGF expression and secretion are increased in the highly aggressive OS cell lines in comparison with non-aggressive OS cell lines.

<p><b>A.</b> Real-time PCR was performed using primers specific for VEGF or β-Actin in the human OS cell lines HOS, MG-63, MNNG/HOS and 143B. Values were obtained from three separate experiments each repeated in triplicate and represent the mean ±S.D. *p<0.01. <b>B.</b> HOS, MG-63, MNNG/HOS and 143B human OS cells were seeded at 1 x 10⁵ cells per well on a 6-well plate. Twenty-four hours later, conditioned media were examined for the presence of human VEGF by ELISA. Values were obtained from three separate experiments each replicated in triplicate and represent the mean ±S.D. *p<0.01. <b>C.</b> HOS, MG-63, MNNG/HOS and 143B human OS cells were seeded at 1 x 10⁵ cells per well on a 6-well plate. Twenty-four hours later, aliquots of supernatant were removed from dishes and added to 12-well plates seeded with 1 x 10⁵ HUVEC cells. Endothelial cell tube-like formation was examined at 12 h. Negative controls of non-conditioned culture media (DMEM + 10% FBS) and conditioned media depleted from VEGF were also included. Representative photomicrographs were taken at 50x magnification from 3 independent experiments, each repeated in triplicate. Scale bar = 50 μm <b>D.</b> Capillary tube-like length was quantified using the ImageJ software. Values were obtained from three separate experiments each repeated in triplicate and represent the mean ±S.D. *p<0.01.</p

Exogenous VEGF partially rescues normal cell phenotype in α-CaMKII silenced 143B OS cells.

<p>143B cells were transduced with lentiviruses expressing either scrambled (shCtrl) or α-CaMKII- targeting shRNAs (shCaMKIIα) <b>A.</b> MTT assay (left panel) was performed at days 1, 2, 3, 4 and 5 to determine the number of viable cells. 5x10³ 143B cells were seeded in a 96-well plate. At 24 hours after seeding, cells were treated with 100nM VEGF. Values were obtained from three separate experiments, each repeated in triplicate and represent the mean ± S.D. *p<0.01. Trypan blue exclusion assay (right panel) was performed at day 5 to determine the live cell number. At 24 hours after seeding, cells were treated with 100nM VEGF every 24 hours. Values were obtained from three separate experiments, each repeated in triplicate and represent the mean ± S.D. *p<0.01. <b>B.</b> Scratch/wound healing assay was performed on cells cultured for 12 hours. The width between the scratched areas at hour 0 was set to 100%. Representative photomicrographs were taken at 50x magnification from 3 independent experiments, each repeated in triplicate. Values represent the mean ± S.D. *p<0.01. <b>C</b>. Trans-well invasion assay allowing cells to invade for 24 hours. Representative photomicrographs were taken at 100x magnifications from 3 independent experiments, each repeated in duplicate. Values represent the mean ± S.D. *p<0.01.</p