Recapitulation of mesenchymal condensation enhances in vitro chondrogenesis of human mesenchymal stem cells

Mesenchymal condensation is a critical transitional stage that precedes cartilage formation during embryonic development. We hypothesized that "priming" hMSCs to recapitulate mesenchymal condensation events prior to inducing differentiation would enhance their subsequent chondrogenic properties. Our prior studies have suggested that exposing hMSCs to hypoxia (2% O(2)) induces condensation-like effects. We therefore assessed the effect of preconditioning for different time periods on the expression of condensation specific genes by growing hMSCs in expansion medium under different normoxic (20% O(2)) and hypoxic conditions for up to 2 weeks, and subsequently induced chondrogenesis of preconditioned hMSCs. The total cultivation time for each group was 4 weeks and the chondrogenic properties were assessed using gene expression, biochemical analysis, and histological staining. Our results demonstrated the benefits of preconditioning were both time- and oxygen-dependent. Condensation specific genes, SOX-9 and NCAM, were significantly up-regulated in hypoxic conditions at the end of 1 week. COL X and MMP13 expression was also lower than the normoxic samples at this time point. However, this group did not exhibit more efficient chondrogenesis after 4 weeks. Instead, hMSCs preconditioned for 1 week and subsequently differentiated, both under 20% O(2), resulted in the most efficient chondrogenesis. Interestingly, while hypoxia appears to positively enhance expression of chondrogenic genes, this did not produce an enhanced matrix accumulation. The results of this study emphasize the significance of considering the timing of specific cues in developing protocols for stem cell-based therapies and underscore the complexity in regulating stem cell differentiation and tissue formation.     

Hypoxic expression of NLRP3 and VEGF in cultured retinal pigment epithelial cells: contribution of P2Y<Subscript>2</Subscript> receptor signaling

Retinal hypoxia is a major condition of the chronic inflammatory disease age-related macular degeneration. Extracellular ATP is a danger signal which is known to activate the NLRP3 inflammasome in various cell systems. We investigated in cultured human retinal pigment epithelial (RPE) cells whether hypoxia alters the expression of inflammasome-associated genes and whether purinergic receptor signaling contributes to the hypoxic expression of key inflammatory (NLRP3) and angiogenic factor (VEGF) genes. Hypoxia and chemical hypoxia were induced by a 0.2%-O₂ atmosphere and addition of CoCl₂, respectively. Gene expression was determined with real-time RT-PCR. Cytosolic NLRP3 and (pro-) IL-1β levels, and the extracellular VEGF level, were evaluated with Western blot and ELISA analyses. Cell culture in 0.2% O₂ induced expression of NLRP3 and pro-IL-1β genes but not of the pro-IL-18 gene. Hypoxia also increased the cytosolic levels of NLRP3 and (pro-) IL-1β proteins. Inflammasome activation by lysosomal destabilization decreased the cell viability under hypoxic, but not control conditions. In addition to activation of IL-1 receptors, purinergic receptor signaling mediated by a pannexin-dependent release of ATP and a release of adenosine, and activation of P2Y₂ and adenosine A₁ receptors, was required for the full hypoxic expression of the NLRP3 gene. P2Y₂ (but not A₁) receptor signaling also contributed to the hypoxic expression and secretion of VEGF. The data indicate that hypoxia induces priming and activation of the NLRP3 inflammasome in cultured RPE cells. The hypoxic NLRP3 and VEGF gene expression and the secretion of VEGF are in part mediated by P2Y₂ receptor signaling.     

Chronic hypoxia promotes an aggressive phenotype in rat prostate cancer cells

In general, tumors cells that are resistant to apoptosis and increase angiogenesis are a result of the hypoxic responses contributing to the malignant phenotype. In this study, we developed a chronic hypoxic cell model (HMLL), by incubating the prostate cancer MatLyLu cells in a hypoxic chamber (1% O₂) over 3 weeks. Surviving cells were selected through each cell passage and were grown in the hypoxic condition up to 8 weeks. This strategy resulted in survival of only 5% of the cells. The surviving hypoxic cells displayed a greater stimulation on hypoxic adaptive response, including a greater expression of glucose transporter1 (Glut1) and VEGF secretion. In addition, higher invasion activity was observed in the chronic hypoxic HMLL cells as compared to MatLyLu cells exposed to acute hypoxia (1% O₂, 5 h) using the matrigel assay. To further examine the role of HIF-1α in tumor progression, both MatLyLu and HMLL cells were transfected with dominant-negative form of HIF-1α (DNHIF-1α). The Matrigel invasion activity induced by chronic hypoxia was significantly attenuated by DNHIF-1α. These results suggest that signaling pathways leading to hypoxic response may be differentially regulated in chronic hypoxic cells and acute hypoxic cells. Chronic hypoxia may play a greater role than acute hypoxia in promoting the aggressive phenotype of tumor cells. This observation mimics the clinical scenario where tumor cells following treatment with radiation are subjected to hypoxic conditions. The reemergence of tumor following treatment usually results in tumor cells that are more aggressive and metastatic.     

Chronic hypoxia promotes an aggressive phenotype in rat prostate cancer cells

In general, tumors cells that are resistant to apoptosis and increase angiogenesis are a result of the hypoxic responses contributing to the malignant phenotype. In this study, we developed a chronic hypoxic cell model (HMLL), by incubating the prostate cancer MatLyLu cells in a hypoxic chamber (1% O(2)) over 3 weeks. Surviving cells were selected through each cell passage and were grown in the hypoxic condition up to 8 weeks. This strategy resulted in survival of only 5% of the cells. The surviving hypoxic cells displayed a greater stimulation on hypoxic adaptive response, including a greater expression of glucose transporter1 (Glut1) and VEGF secretion. In addition, higher invasion activity was observed in the chronic hypoxic HMLL cells as compared to MatLyLu cells exposed to acute hypoxia (1% O(2), 5 h) using the matrigel assay. To further examine the role of HIF-1alpha in tumor progression, both MatLyLu and HMLL cells were transfected with dominant-negative form of HIF-1alpha (DNHIF-1alpha). The Matrigel invasion activity induced by chronic hypoxia was significantly attenuated by DNHIF-1alpha. These results suggest that signaling pathways leading to hypoxic response may be differentially regulated in chronic hypoxic cells and acute hypoxic cells. Chronic hypoxia may play a greater role than acute hypoxia in promoting the aggressive phenotype of tumor cells. This observation mimics the clinical scenario where tumor cells following treatment with radiation are subjected to hypoxic conditions. The reemergence of tumor following treatment usually results in tumor cells that are more aggressive and metastatic.     

Prolyl-hydroxylase inhibition and HIF activation in osteoblasts promotes an adipocytic phenotype

Bone is a dynamic environment where cells sense and adapt to changes in nutrient and oxygen availability. Conditions associated with hypoxia in bone are also associated with bone loss. In vitro hypoxia (2% oxygen) alters gene expression in osteoblasts and osteocytes and induces cellular changes including the upregulation of hypoxia inducible factor (HIF) levels. Our studies show that osteoblasts respond to hypoxia (2% oxygen) by enhancing expression of genes associated with adipocyte/lipogenesis phenotype (C/EBPbeta, PPARgamma2, and aP2) and by suppressing expression of genes associated with osteoblast differentiation (alkaline phosphatase, AP). Hypoxia increased HIF protein levels, hypoxic response element (HRE) binding, and HRE-reporter activity. We also demonstrate that prolyl-hydroxylases 2 and 3 (PHD2, PHD3), one of the major factors coordinating HIF degradation under normoxic but not hypoxic conditions, are induced in osteoblasts under hypoxic conditions. To further determine the contribution of PHDs and upregulated HIF activity in modulating osteoblast phenotype, we treated osteoblasts with a PHD inhibitor, dimethyloxaloylglycine (DMOG), and maintained cells under normoxic conditions. Similar to hypoxic conditions, HRE reporter activity was increased and adipogenic gene expression was increased while osteoblastic genes were suppressed. Taken together, our findings indicate a role for PHDs and HIFs in the regulation of osteoblast phenotype.     

Expression and hypoxia-responsiveness of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4 in mammary gland malignant cell lines

Recently, we have shown that PFKFB4 gene which encodes the testis isoenzyme of PFKFB is also expressed in the prostate and hepatoma cancer cell lines. Here we have studied expression and hypoxic regulation of the testis isoenzyme of PFKFB4 in several malignant cell lines from a female organ--the mammary gland. Our studies clearly demonstrated that PFKFB4 mRNA is also expressed in mammary gland malignant cells (MCF-7 and T47D cell lines) in normoxic conditions and that hypoxia strongly induces it expression. To better understand the mechanism of hypoxic regulation of PFKFB4 gene expression, we used dimethyloxalylglycine, a specific inhibitor of HIF-1alpha hydroxylase enzymes, which strongly increases HIF-1alpha levels and mimics the effect of hypoxia. It was observed that PFKFB4 expression in the MCF7 and T47D cell lines was highly responsive to dimethyloxalylglycine, suggesting that the hypoxia responsiveness of PFKFB4 gene in these cell lines is regulated by HIF-1 proteins. Moreover, desferrioxamine and cobalt chloride, which mimic the effect of hypoxia by chelating or substituting for iron, had a similar stimulatory effect on the expression of PFKFB mRNA. In other mammary gland malignant cell lines (BT549, MDA-MB-468, and SKBR-3) hypoxia and hypoxia mimics also induced PFKFB4 mRNA, but to variable degrees. The hypoxic induction of PFKFB4 mRNA was equivalent to the expression of PFKFB3, Glut1, and VEGF, which are known HIF-1-dependent genes. Hypoxia and dimethyloxalylglycine increased the PFKFB4 protein levels in all cell lines studied except MDA-MB-468. Through site-specific mutagenesis in the 5'-flanking region of PFKFB4 gene the hypoxia response could be limited. Thus, this study provides evidence that PFKFB4 gene is also expressed in mammary gland cancer cells and strongly responds to hypoxia via an HIF-1alpha dependent mechanism. Moreover, the PFKFB4 and PFKFB3 gene expression in mammary gland cancer cells has also a significant role in the Warburg effect which is found in all malignant cells.