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. / published_or_final_version / Orthopaedics and Traumatology / Doctoral / Doctor of Philosophy
| Identifer | oai:union.ndltd.org:HKU/oai:hub.hku.hk:10722/174535 |
| Date | January 2012 |
| Creators | Tam, Wai-kit., 譚偉傑. |
| Contributors | Cheung, KMC, Leung, YLV, Chan, D |
| Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
| Source Sets | Hong Kong University Theses |
| Language | English |
| Detected Language | English |
| Type | PG_Thesis |
| Source | http://hub.hku.hk/bib/B47849770 |
| Rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works., Creative Commons: Attribution 3.0 Hong Kong License |
| Relation | HKU Theses Online (HKUTO) |
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