Cathepsin V Research Articles

Decreased cathepsin V expression due to Fli1 deficiency contributes to the development of dermal fibrosis and proliferative vasculopathy in systemic sclerosis

Cathepsin V (CTSV) is a proteolytic enzyme potentially modulating angiogenic processes, collagen degradation and keratinocyte differentiation. We aimed to investigate the clinical association of serum CTSV levels and the mechanism by which CTSV expression is altered in SSc. Serum CTSV levels were determined by ELISA in 51 SSc and 18 healthy subjects. CTSV expression was evaluated by immunostaining in SSc and normal skin and by RT-real-time PCR in normal and SSc dermal fibroblasts, normal dermal fibroblasts treated with TGF-β1 or Fli1 siRNA and human dermal microvascular endothelial cells (ECs) treated with Fli1 siRNA. Serum CTSV levels were significantly lower in dcSSc and lcSSc patients than in healthy controls. In early-stage dcSSc, serum CTSV levels were remarkably and uniformly decreased compared with healthy controls. The decrease in serum CTSV levels in mid- and late-stage dcSSc and in lcSSc was linked to the development of proliferative vasculopathy. CTSV expression was decreased in microvascular ECs, pericytes/vascular smooth muscle cells and keratinocytes of dcSSc and lcSSc skin and in dermal fibroblasts of dcSSc skin compared with control skin. Consistently, CTSV expression was decreased in cultured dermal fibroblasts from early-stage dcSSc. Furthermore, mRNA levels of the CTSV gene were significantly decreased in normal fibroblasts treated with TGF-β1 or Fli1 siRNA and in human dermal microvascular ECs treated with Fli1 siRNA. Loss of CTSV expression may contribute to the development of fibrosis, vasculopathy and the altered phenotype of keratinocytes in SSc.

Colocalization of Cystatin M/E and Cathepsin V in Lamellar Granules and Corneodesmosomes Suggests a Functional Role in Epidermal Differentiation

Cystatin M/E is a cysteine protease inhibitor with two distinct binding sites for papain-like cysteine proteases (family C1) and the asparaginyl endopeptidase (AEP) legumain of family C13. We have previously demonstrated that deficiency of cystatin M/E in mice causes ichthyosiform skin changes and barrier disruption, which could be caused by unrestrained AEP activity. Recently, we provided biochemical evidence that human cathepsin V (CTSV) and cathepsin L (CTSL) are additional biological targets for human cystatin M/E. To address the possible role of these three proteases and their inhibitor in epidermal differentiation, we investigated the localization of these proteins in normal human skin. Whereas CTSL and AEP were broadly expressed in epithelial cells of the skin, we found a specific colocalization of cystatin M/E and CTSV in the stratum granulosum and in the root sheets of the hair follicle, using immunofluorescence microscopy. Immunoelectron microscopy revealed that cystatin M/E and CTSV are separately transported within the lamellar granules. Cystatin M/E was also found in the extracellular space in the stratum corneum associated with corneodesmosomes, where it was closely associated with CTSV. Based on the striking stratum-specific colocalization of cystatin M/E and CTSV, we propose that these molecules could have an important role in epidermal differentiation and desquamation.

Cystatin M/E Is a High Affinity Inhibitor of Cathepsin V and Cathepsin L by a Reactive Site That Is Distinct from the Legumain-binding Site: A NOVEL CLUE FOR THE ROLE OF CYSTATIN M/E IN EPIDERMAL CORNIFICATION

Cystatin M/E is a high affinity inhibitor of the asparaginyl endopeptidase legumain, and we have previously reported that both proteins are likely to be involved in the regulation of stratum corneum formation in skin. Although cystatin M/E contains a predicted binding site for papain-like cysteine proteases, no high affinity binding for any member of this family has been demonstrated so far. We report that human cathepsin V (CTSV) and human cathepsin L (CTSL) are strongly inhibited by human cystatin M/E. Kinetic studies show that Ki values of cystatin M/E for the interaction with CTSV and CTSL are 0.47 and 1.78 nM, respectively. On the basis of the analogous sites in cystatin C, we used site-directed mutagenesis to identify the binding sites of these proteases in cystatin M/E. We found that the W135A mutant was rendered inactive against CTSV and CTSL but retained legumain-inhibiting activity. Conversely, the N64A mutant lost legumain-inhibiting activity but remained active against the papain-like cysteine proteases. We conclude that legumain and papain-like cysteine proteases are inhibited by two distinct non-overlapping sites. Using immunohistochemistry on normal human skin, we found that cystatin M/E co-localizes with CTSV and CTSL. In addition, we show that CTSL is the elusive enzyme that processes and activates epidermal transglutaminase 3. The identification of CTSV and CTSL as novel targets for cystatin M/E, their (co)-expression in the stratum granulosum of human skin, and the activity of CTSL toward transglutaminase 3 strongly imply an important role for these enzymes in the differentiation process of human epidermis.