Abstract
Degradation of extracellular matrix (ECM) during tendinopathy is, in part, mediated by the collagenolytic cathepsin K (catK) and cathepsin L (catL), with a temporal component to their activity. The objective of this study was to determine how catK and catL act in concert or in conflict to degrade collagen and tendon ECM during tissue degeneration. To do so, type I collagen gels or ECM extracted from apolipoprotein E deficient mouse Achilles tendons were incubated with catK and catL either concurrently or sequentially, incubating catK first, then catL after a delayed time period. Sequential incubation of catK then catL caused greater degradation of substrates over concurrent incubation, and of either cathepsin alone. Zymography showed there were reduced amounts of active enzymes when co-incubated, indicating that cannibalism, or protease-on-protease degradation between catK and catL was occurring, but incubation with ECM could distract from these interactions. CatK alone was sufficient to quickly degrade tendon ECM, but catL was not, requiring the presence of catK for degradation. Together, these data identify cooperative and conflicting actions of cathepsin mediated collagen matrix degradation by considering interactive effects of multiple proteases during tissue degeneration.
Highlights
In previous studies, we demonstrated upregulated active cathepsins in a treadmill-based rat model of supraspinatus overuse injury[7,21]
The amount of collagen cleaved is expressed as percent of collagen fragments present in the Coomassie stained polyacrylamide gel quantified with densitometry, normalized to control conditions of collagen gel or tendon extracellular matrix (ECM) incubated in the absence of any proteases over the 8 hr period
After normalizing to the no-cathepsin control that is equivalent to the pellet fractions of the enzyme incubated conditions, 93% of the collagen chains were cleaved in the sequential condition, significantly more than when cathepsin K (catK) or cathepsin L (catL) were incubated with collagen gels alone (29% and 37% cleaved, respectively) or co-incubated with collagen gels concurrently (55% cleaved) (Fig. 1B, n = 3, p < 0.05)
Summary
We demonstrated upregulated active cathepsins in a treadmill-based rat model of supraspinatus overuse injury[7,21]. CatK is subject to autodigestion, or self-cleavage over time[37], and we have demonstrated that cathepsins can hydrolyze other cathepsins, even in the presence of a substrate, a phenomenon we termed cathepsin cannibalism[37,38] These studies suggest that cooperative substrate cleavage, autodigestion, and cannibalism among multiple proteolytic enzymes can dictate the amount of active proteases in a system, impacting rate and amount of tissue degradation. It is still unclear how multiple cathepsins, present simultaneously, impact tendon ECM degradation, type I collagen. These experimental systems were used to test the hypothesis that catK initiation of substrate degradation primes the substrates by exposing sites susceptible to further hydrolysis by catL, and pre-incubating matrix substrate with catK before introducing catL, resulting in greater hydrolysis than when concurrently co-incubated with catK and catL
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
