HIF-1α controls extracellular matrix synthesis by epiphyseal chondrocytes

Abstract

The transcription factor HIF-1α plays a crucial role in modifying gene expression during low oxygen tension. In a previous study, we demonstrated that HIF-1α is essential for chondrocyte growth arrest and survival in vivo. To explore further the role of HIF-1α in cartilage biology, we undertook studies with primary epiphyseal chondrocytes with a targeted deletion of HIF-1α. In this study, we show that HIF-1α is necessary for regulating glycolysis under aerobic and anaerobic conditions. HIF-1α-null chondrocytes were unable to maintain ATP levels in hypoxic microenvironments, indicating a fundamental requirement for this factor for the regulation of chondrocyte metabolism. Synthesis of the angiogenic factor vascular endothelial growth factor was also significantly induced by hypoxia,and this increase is lost in HIF-1α-null mutant cells. Under hypoxic conditions, aggrecan mRNA and protein levels were significantly reduced in chondrocytes lacking the HIF-1α transcription factor. Interestingly,strongly increased type-II collagen protein levels were detected in wild-type cells after 44 hours of hypoxia. In addition, type-II collagen mRNA and protein levels were strongly decreased under low oxygen in chondrocytes lacking HIF-1α. In summary, our results clearly demonstrate the importance of HIF-1α in maintenance of anaerobic glycolysis, and thereby extracellular matrix synthesis, of epiphyseal chondrocytes.

Deletion ofVhlhin chondrocytes reduces cell proliferation and increases matrix deposition during growth plate development

“…Interestingly, viable chondrocytes at the periphery display a significant increased rate of actively proliferating cells. Furthermore, we have provided clear evidence that HIF1α is necessary for type II collagen accumulation by hypoxic growth plate chondrocytes in vitro (Pfander et al, 2003). Based on these findings, we hypothesized pVHL, a key molecule in the degradation pathway of HIF1α, to be an important modulator of long bone development, possibly through the regulation of HIF1α stability.…”

“…Detection of binding was by the Streptdavidin-HRP system provided by the TSA kit (Perkin Elmer-Life Sciences, MA, USA), according to the manufacturer's conditions. Chondrocyte isolation, western blot analysis, real-time PCR, Elisa Chondrocytes were isolated from newborn Vhlh +f//+f mice as described (Pfander et al, 2003). In brief, forelimbs and hindlimbs were dissected, and distal epiphyses of radius, ulna and tibia were isolated in HBSS (Gibco BRL, MD, USA).…”

“…Total RNA was isolated from wild-type chondrocytes or Vhlh null cells as previously described (Pfander et al, 2003), and then transcribed into single cDNA using AMV reverse transcriptase (Boehringer Mannheim, Germany) and random hexamer primers, according to the manufacturer's instructions. For real-time PCR analyses, cDNA was diluted to a final concentration of 10 ng/µl.…”

Hypoxia‐inducible factor 2α is essential for hypoxic induction of the human articular chondrocyte phenotype

“…Pfander and colleagues cultured HIF-1␣-null and wild-type murine epiphyseal chondrocytes in normoxia and in hypoxia, in order to investigate the contribution of HIF-1␣ to cell metabolism (16). Phosphoglycerate kinase (PGK), GLUT1, and VEGF were all clearly up-regulated by hypoxia in wild-type cells but not in HIF-1␣-null cells, indicating the importance of HIF-1␣ for hypoxic induction of these genes.…”

“…HIF-1␣ was also implicated in relation to matrix synthesis by isolated murine epiphyseal chondrocytes (16) and human osteoarthritis chondrocytes (17). However, the role of HIF-1␣ in hypoxic induction of the human articular chondrocyte phenotype remains uncertain, while that of HIF-2␣ is completely unexplored.…”