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

TitleRole of hypoxia inducible factor-alpha (HIF-α) genes inchondrogenesis
Authors
Advisors
Issue Date2012
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Tam, W. [譚偉傑]. (2012). Role of hypoxia inducible factor-alpha (HIF-α) genes in chondrogenesis. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b4784977
AbstractCartilage 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.
DegreeDoctor of Philosophy
SubjectAnoxemia.
Chondrogenesis.
Dept/ProgramOrthopaedics and Traumatology

 

DC FieldValueLanguage
dc.contributor.advisorCheung, KMC-
dc.contributor.advisorLeung, YLV-
dc.contributor.advisorChan, D-
dc.contributor.authorTam, Wai-kit.-
dc.contributor.author譚偉傑.-
dc.date.issued2012-
dc.identifier.citationTam, W. [譚偉傑]. (2012). Role of hypoxia inducible factor-alpha (HIF-α) genes in chondrogenesis. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b4784977-
dc.description.abstractCartilage 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.-
dc.languageeng-
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)-
dc.relation.ispartofHKU Theses Online (HKUTO)-
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.-
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License-
dc.source.urihttp://hub.hku.hk/bib/B47849770-
dc.subject.lcshAnoxemia.-
dc.subject.lcshChondrogenesis.-
dc.titleRole of hypoxia inducible factor-alpha (HIF-α) genes inchondrogenesis-
dc.typePG_Thesis-
dc.identifier.hkulb4784977-
dc.description.thesisnameDoctor of Philosophy-
dc.description.thesislevelDoctoral-
dc.description.thesisdisciplineOrthopaedics and Traumatology-
dc.description.naturepublished_or_final_version-
dc.identifier.doi10.5353/th_b4784977-
dc.date.hkucongregation2012-

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