Role of hypoxia inducible factor-alpha (HIF-α) genes inchondrogenesis

Abstract

Cartilage is an essential skeletal connective tissue in vertebrates. It comprises

extracellular matrix components, especially for collagens and proteoglycans. Once

the cartilage is damaged, it has limited self-repair capacity. Thus, by

understanding the dynamic cellular process of chondrogenesis and chondrocyte

differentiation would be necessary in developing therapeutic approaches for

cartilage repair. Currently, there is a great interest in the development of cell

therapy to repair damaged tissues. In particularly, human mesenchymal stem cells

(hMSCs) are attractive candidates for the treatment of skeletal system disorders

because they can be greatly expanded ex vivo and they readily differentiate into

chondrocytes upon stimulation. Studies have demonstrated low environmental

oxygen tension could affect the chondrogenic differentiation of hMSCs. The three

basic helix-loop-helix (bHLH) motif-containing hypoxia inducible factor α (HIF-α)

subunits (i.e. HIF-1α, HIF-2α and HIF-3α) are the major oxygen-sensitive

transcription factors regulating physiological responses under hypoxia. Of

significance, HIF-1α has been reported to induce a hyaline chondrocyte-like

phenotype in human articular chondrocytes. The aim of this study was to

investigate the roles of all three human HIF-α paralogues in chondrogenesis,

particularly for the transcriptional regulation of chondrocyte-specific genes,

including type II collagen (COL2A1) and aggrecan (AGC1). The effect of all three

human HIF-α paralogues on the chondrogenic differentiation of hMSCs could

then be investigated. Self-inactivating lentivirus vector (SIN-LV) shuttle plasmids

coding for murine SOX9, wild-type and oxygen-insensitive versions of human

HIF-1α and HIF-2α or wild-type HIF-3α were generated. These plasmids were

used in luciferase-based promoter assays and to generate SIN-LV particles for

overexpression studies. Our data revealed that SOX9, a key transactivator of

chondrogenesis, strongly activates the transcription of COL2A1 and AGC1.

Ectopic expression of HIF-2α could also induce the transcription of COL2A1 and

AGC1. Strikingly, a cooperative transcriptional up-regulation of COL2A1 and

AGC1 via the overexpression of HIF-1α and SOX9 was observed. Furthermore,

HIF-3α was shown to inhibit the SOX9–dependent transcriptional up-regulation

of COL2A1 and AGC1. Here, the multipotency of hMSCs cultured under

hypoxia (1% O2 tension) was also illustrated. A pilot study for overexpressing

exogenous gene in chondrogenic stimulated hMSC pellets via SIN-LV particles is

described. Eventually, a rationale is provided for manipulating HIF-α expression

in chondrogenic stimulated hMSC pellet via SIN-LVs encoding HIF-α subunits to

study the contribution of HIF-α paralogues on promoting the chondrogenic

differentiation of hMSCs.