Kallikrein-related Peptidase 12 Hydrolyzes Matricellular Proteins of the CCN Family and Modifies Interactions of CCN1 and CCN5 with Growth Factors

Abstract

Kallikrein-related peptidases (KLKs) are an emerging group of secreted serine proteases involved in several physiological and pathological processes. We used a degradomic approach to identify potential substrates of KLK12. MDA-MB-231 cells were treated either with KLK12 or vehicle control, and the proteome of the overlying medium was analyzed by mass spectrometry. CCN1 (cyr61, ctgf, nov) was among the proteins released by the KLK12-treated cells, suggesting that KLK12 might be responsible for the shedding of this protein from the cell surface. Fragmentation of CCN1 by KLK12 was further confirmed in vitro, and the main cleavage site was localized in the hinge region between the first and second half of the recombinant protein. KLK12 can target all six members of the CCN family at different proteolytic sites. Limited proteolysis of CCNs (cyr61, ctgf, nov) was also observed in the presence of other members of the KLK family, such as KLK1, KLK5, and KLK14, whereas KLK6, KLK11, and KLK13 were unable to fragment CCNs. Because KLK12 seems to have a role in angiogenesis, we investigated the relations between KLK12, CCNs, and several factors known to be involved in angiogenesis. Solid phase binding assays showed that fragmentation of CCN1 or CCN5 by KLK12 prevents VEGF 165 binding, whereas it also triggers the release of intact VEGF and BMP2 from the CCN complexes. The KLK12-mediated release of TGF-␤1 and FGF-2, either as intact or truncated forms, was found to be concentration-dependent. These findings suggest that KLK12 may indirectly regulate the bioavailability and activity of several growth factors through processing of their CCN binding partners.

Degradome Products of the Matricellular Protein CCN1 as Modulators of Pathological Angiogenesis in the Retina

“…Indeed, the primary sequence of CCN1 contains numerous cleavage sites for aspartic, cysteine, and serine proteases. Concordantly, Guillon-Munos et al (46) have validated CCN1 as a substrate of kallikreinrelated peptidases, an emerging group of secreted serine proteases that cleave CCN1 at different sites and regulate angiogenesis. Similarly, plasmin, a serine protease that promotes fibrinolysis, cleaves CCN1 either directly or indirectly via activation of MMPs (47).…”

CCN1 secretion and cleavage regulate the lung epithelial cell functions after cigarette smoke

“…The majority of flCCN1 or cCCN1 was detected in exosome or the soluble component, respectively (Fig. 1H), suggesting a differential route of secretion for flCCN1 or cCCN1 after CSE stimulation despite that CCN1 cleavage has been reported in breast cancer tissue previously (14,37). CS-induced CCN1 cleavage in lung tissue and its functional roles in lung diseases remain unexplored.…”

Kallikrein-related peptidases in prostate, breast, and ovarian cancers: from pathobiology to clinical relevance

“…A more recent study proposed an alternative molecular pathway for the explanation of KLK12-stimulated angiogenesis. KLK12 was found to cleave the CCN1 and CCN5 matricellular proteins and thus to regulate the bioavailability of VEGF, bone morphogenetic protein 2 (BMP2), TGF β 1 and FGF2 angiogenesis-promoting growth factors (Guillon -Munos et al, 2011 ). The fact that KLK12 is up-regulated in prostate cancer patients allows us to hypothesize the in vivo angiogenic role of KLK12 in prostate malignancy.…”