Background/Purpose: Recently Mohawk homeobox (MKX) has been discovered as a tendon and ligament specific transcriptional factor. MKX deficient-mice are shown to hypoplastic tendons throughout the body by down regulating changing type I collagen production in tendon cells. However, in human tendons or ligaments, little is known about the function of MKX. In this study, we demonstrated using human anterior cruciate ligament (ACL) tissues and primary cultured human ACL cells that MKX is an important regulator of human ligament homeostasis, and that its changes in the expression and function play an important role in ligament degeneration. The aims of this study are to characterize the expression of MKX in normal human knee joints and osteoarthritis (OA) affected knee joints and to investigate the role of MKX in human ligament homeostasis.

Methods: Human ACL specimens were obtained from the knee joints at cadaveric autopsy within 24-48 hours postmortem with approval of the Scripps Human Subjects Committee. 7 normal donors (mean±SD age 33.57±11.67years) and 8 donors with OA (mean±SD age 77.50±11.46 years) were analyzed and none of the donors had a history of knee joint trauma. All cartilage surfaces were graded macroscopically. ACL degeneration was assessed macroscopically and histologically using quantitative scoring systems. ACL tissues were analyzed for the expression of MKX by immunohistochemistry and quantitative RT-PCR assays. Primary cultured human ACL cells were stimulated with IL-1β to examine whether pro-inflammatory cytokine modulates MKX expression. Moreover, in order to examine the function of MKX on ECM production and differentiation in these cells, we performed the knocked-down by MKX specific siRNA and then, determined the expression of some genes involving in ECM and the differentiation using quantitative RT-PCR and western blotting analysis. 

Results: The expression of MKX was remarkably decreased during developing OA. In addition, the expression of COL1a1, which is major component of ACL, is also decreased in OA group. In coincidence with the result of immunohistochemistry revealed that the percentage of MKX positive cells was significantly reduced in OA group. In primary cultured human ACL cells, the expression of MKX was significantly reduced by the treatment of IL-1β. The expression of genes involving in regulating ECM homeostasis such as COL1a1 and TNXb and transcriptional factor Scleraxis (SCX), which is specific regulator of the tendon/ligament lineage, were down-regulated by IL-1β treatment. On the other hand, SOX9, which is involved in the modulation of chondrocyte like phenotype, was up-regulated by IL-1β treatment. The expression of COL1a1 and TNXb were decreased by MKX specific siRNA treatment, though that of SOX9 was increased. We didn’t detect any significant differences in those of SCX, IL-6 and MMP13.

Conclusion: To our knowledge, this is the first report to investigate the MKX expression and function in Human ligaments. The present study demonstrates that the expression of MKX is down-regulated by an inflammatory response and MKX may play important roles in ligament homeostasis via regulating the expression of COL1a1, TNXb and SOX9 in human ACL cells.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-transcription-factor-mohawk-plays-an-important-role-for-maintaining-human-anterior-cruciate-ligament-homeostasis/