Background/Purpose:  We recently reported changes in gene expression in knee joint tissue from 12 week-old and 12 month-old mice after OA was induced by surgical destabilization of the medial meniscus (DMM). Heparin-binding EGF-like growth factor (HB-EGF) was found by gene array to be significantly up-regulated at 8 weeks after surgery in both age groups. HB-EGF is a known ligand for the EGF receptor (EGFR) which signals through MAP kinases and the small GTPase Rac. EGFR signaling has been implicated in OA pathogenesis but a potential role for HB-EGF has not been examined. The purpose of this study was to determine if HB-EGF was present in human OA cartilage and to test the ability of HB-EGF to stimulate production of chondrocyte MMP-13 which can contribute to matrix destruction in OA.

Methods:  Real-time PCR was used to confirm the HB-EGF gene array results using RNA isolated from knee joint tissues (n=9 mice per surgical group) from 12 week-old and 12 month-old male C57/BL6 mice that had undergone DMM or sham control surgery. Immunohistochemistry for HB-EGF was performed using knee joint tissue collected from normal tissue donors or age-matched patients who had undergone knee replacement for OA (n=4 each). In vitro stimulation studies used cultured human articular chondrocytes isolated from normal donor tissue (n≥3 independent donors for each experiment). Fibronectin fragments (FN-f) were used as a catabolic stimulus relevant to OA. Effects of recombinant HB-EGF, ± FN-F, on MMP-13 production were measured by immunoblotting and real time (RT)-PCR and on Rac activity using an activity assay.    

Results:  HB-EGF expression was about two-thirds lower (p<0.05) in the sham control joints of the older mice compared to young controls but increased by >6-fold (p<0.001) in the older DMM joints vs 1.4-fold in the young DMM when compared to the age-matched sham. Immunohistochemical staining revealed increased HB-EGF in human OA cartilage relative to age-matched normal where little to no staining was observed. Treatment of cultured human chondrocytes with FN-f increased HB-EGF expression which peaked at almost 60-fold (by RT-PCR) at 3 hours. Overnight FN-f stimulation increased HB-EGF secretion into conditioned media. We found that HB-EGF at 10ng/ml stimulated chondrocyte Rac activity and phosphorylation of the EGFR, ERK and p38 but not JNK. High dose (100ng/ml) but not low dose (10ng/ml) HB-EGF stimulated MMP-13 production in conditioned media, detected by immunoblotting. However, low dose HB-EGF enhanced the MMP-13 production induced by FN-F by 1.4-fold over FN-f alone and this was completely inhibited by blocking Rac1 with 100µM NSC23766 or EGFR with 250 nM AG1478.

Conclusion: HB-EGF was detected in human OA cartilage but not normal cartilage. FN-fs, known to be present in OA cartilage and to stimulate catabolic pathways, were found to stimulate HB-EGF production by normal chondrocytes. Given the finding that HB-EGF signals through the chondrocyte EGFR and promotes MAP kinase and MMP-13 production and that it is present in human OA cartilage, further evaluation of its role in OA is warranted.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/heparin-binding-epidermal-growth-factor-like-growth-factor-hb-egf-as-a-potential-mediator-in-osteoarthritis/