Oxygen Tension Affects Lubricin Expression in Chondrocytes

Abstract

We assessed the effects of oxygen tension on lubricin expression in bovine chondrocytes and cartilage explants and a role for hypoxia-inducible transcription factor (HIF)-1α in regulating lubricin expression was investigated using a murine chondroprogenitor cell line, ATDC5, and bovine chondrocytes isolated from superficial and middle/deep zones of femoral cartilage. ATDC5 cells and bovine chondrocytes were cultured in micromass under different oxygen tensions (21%, 5%, and 1%). ATDC5 cells and middle/deep zone chondrocytes that initially had low lubricin expression levels were also cultured with or without transforming growth factor (TGF)-β1. Quantitative reverse transcription (RT)-PCR was used to determine lubricin and chondrogenic marker gene mRNA levels and immunohistochemistry was used to assess lubricin protein expression. Explant cartilage plugs cultured under different oxygen tensions were also subjected to immunohistological analysis for lubricin. HIF-1α gene silencing was achieved by electroporatic transfer into ATDC5 cells. A low oxygen tension reduced lubricin gene expression levels in bovine superficial chondrocytes, TGF-β1-treated middle/deep zone chondrocytes, and TGF-β1-treated ATDC5 cells. Lubricin expression in explant cartilage was also suppressed under hypoxia. HIF-1α gene silencing in ATDC5 cells attenuated the lubricin expression response to the oxygen tension. These results corroborate with previous studies that the oxygen tension regulates lubricin gene expression and suggest that HIF-1α plays an important role in this regulation. The normal distribution of lubricin in articular cartilage may be due to the hypoxic oxygen environment of cartilage as it is an avascular tissue. An oxygen tension gradient may be a key factor for engineering cartilage tissue with a layered morphology.

Responses to altered oxygen tension are distinct between human stem cells of high and low chondrogenic capacity

“…HIF-1α has also been reported to activate transcription of LOX —the gene coding for lysyl oxidase, which is an enzyme that catalyzes collagen crosslinking in articular cartilage through a hypoxia-responsive element (HRE) [ 48 ], but only ACPs demonstrated a significant upregulation of LOX . Conclusions regarding the regulation of PRG4 , the gene for lubricin, in response to alterations in oxygen vary in the literature, even though PRG4 also contains an HRE in the promoter region [ 45 , 49 , 50 ]. We have shown here that highly chondrogenic cells upregulated PRG4 gene expression in physioxia.…”

Repair of Damaged Articular Cartilage: Current Approaches and Future Directions

“…Low levels of oxygen also slow age-related changes in the composition and structure of the ECM [178]. However, the effect of hypoxia on the expression of PRG4 by superficial cells is rather controversial [179,180]. Assuming that oxygen is supplied to the joint predominantly via synovial fluid, the superficial zone should be exposed to the highest levels, and indeed, a gradient of oxygen tension exists across the layers of cartilage [178].…”

Hypoxia Inducible Factor-1α in Osteochondral Tissue Engineering

“…The cellular response to oxygen plays an important role in both the development and maintenance of OC tissue [2][3][4][5][6][7] and is primarily mediated through the HIF pathway ( Fig. 1).…”