Abstract
Several members of the ADAMTS (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) family have been identified as aggrecanases, whose substrates include versican, the principal large proteoglycan in the tendon extracellular matrix. We have characterized the expression of ADAMTS-4 in human Achilles tendon and tendon-derived cells. ADAMTS-4 mRNA levels were higher in ruptured tendon compared with normal tendon or chronic painful tendinopathy. In tissue extracts probed by Western blotting, mature ADAMTS-4 (68 kDa) was detected only in ruptured tendons, while processed ADAMTS-4 (53 kDa) was detected also in chronic painful tendinopathy and in normal tendon. In cultured Achilles tendon cells, transforming growth factor-β (TGF-β) stimulated ADAMTS-4 mRNA expression (typically 20-fold after 24 h), while interleukin-1 induced a smaller, shorter-term stimulation which synergised markedly with that induced by TGF-β. Increased levels of immunoreactive proteins consistent with mature and processed forms of ADAMTS-4 were detected in TGF-β-stimulated cells. ADAMTS-4 mRNA was expressed at higher levels by tendon cells in collagen gels than in monolayer cultures. In contrast, the expression of ADAMTS-1 and -5 mRNA was lower in collagen gels compared with monolayers, and these mRNA showed smaller or opposite responses to growth factors and cytokines compared with that of ADAMTS-4 mRNA. We conclude that both ADAMTS-4 mRNA and ADAMTS-4 protein processing may be differentially regulated in normal and damaged tendons and that both the matrix environment and growth factors such as TGF-β are potentially important factors controlling ADAMTS aggrecanase activities in tendon pathology.
Highlights
Tendons such as the Achilles are susceptible to chronic painful degenerative tendinopathy (Åström and Rausing, 1995) and Abbreviations: ADAMTS, A Disintegrin And Metalloproteinase with ThromboSpondin motifs; Ct, threshold cycle; DMEM, Dulbecco's modified Eagle's medium; Epidermal growth factor (EGF), epidermal growth factor; FCS, fetal calf serum; FGF, fibroblast growth factor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IL-1β, interleukin-1β; plateletderived growth factor (PDGF), platelet-derived growth factor; TGF-β, transforming growth factor-β; TNF, tumour necrosis factor
Various studies have indicated that there is a balance between the synthesis and breakdown of matrix components in normal tendons, and that this balance is disrupted in tendinopathies
In this paper we have shown that mRNA encoding the aggrecanase ADAMTS-4 is regulated independently from ADAMTS-1 or ADAMTS-5 mRNA in both human Achilles tendon tissue samples and cultured tendon cells, and that mature and processed forms of ADAMTS-4 protein may be detected in tendon tissue and cellular extracts
Summary
Tendons such as the Achilles are susceptible to chronic painful degenerative tendinopathy (Åström and Rausing, 1995) and Abbreviations: ADAMTS, A Disintegrin And Metalloproteinase with ThromboSpondin motifs; Ct, threshold cycle; DMEM, Dulbecco's modified Eagle's medium; EGF, epidermal growth factor; FCS, fetal calf serum; FGF, fibroblast growth factor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IL-1β, interleukin-1β; PDGF, platelet-derived growth factor; TGF-β, transforming growth factor-β; TNF, tumour necrosis factor. Using relative quantitative RT-PCR, we recently screened RNA samples from normal Achilles tendons and from individuals suffering either chronic tendon pain or spontaneous tendon rupture, for mRNA encoding all the known matrix metalloproteinases, ADAMTS and tissue inhibitors of metalloproteinases (TIMP) (Jones et al, 2006). MRNA encoding the aggrecanases ADAMTS-1, -4, -5, -8, -9 and -15 were each detected Of these mRNA, ADAMTS-1 mRNA was expressed at highest levels but showed little difference between normal, painful and ruptured tendons, while ADAMTS4 mRNA showed the largest change, increasing 8-fold in ruptured tendons (Jones et al, 2006). Dimensional collagen gel cultures, and examined the expression of ADAMTS-4 protein in tendon tissues and cells
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