Schechter N M, Brass L F, Lavker R M, Jensen P J
Department of Dermatology, University of Pennsylvania, Philadelphia 19104-6142, USA.
J Cell Physiol. 1998 Aug;176(2):365-73. doi: 10.1002/(SICI)1097-4652(199808)176:2<365::AID-JCP15>3.0.CO;2-2.
Protease activated receptors (PARs) compose a family of G protein signal transduction receptors activated by proteolysis. In this study, the susceptibility of PARs expressed on human keratinocytes and dermal fibroblasts to the human mast cell proteases tryptase and chymase was evaluated. PAR activation was measured by monitoring cytosolic [Ca2+] in cells loaded with the fluorescent Ca2+ probe Fura-2. Tryptase produced transient cytosolic Ca2+ mobilization in keratinocytes, but not in fibroblasts. Ca2+ mobilization in keratinocytes required enzymatically active tryptase, demonstrated desensitization, and was blocked by pretreatment of cells with the PAR-2 peptide agonist SLIGKV, trypsin, or the phospholipase inhibitor U73122. Heparin, a GAG that binds to tryptase, stabilizing its functional form, also inhibited tryptase-induced Ca2+ mobilization. The maximal response elicited by tryptase was smaller than that observed upon treatment of keratinocytes with trypsin, a known activator of PAR-2, and keratinocytes made refractory to tryptase by pretreatment with the protease remained responsive to trypsin. Pretreatment of keratinocytes with thrombin, an activator of PAR-1 and -3 (thrombin receptors), had no detectable effect on the tryptase or trypsin responses. These data suggest that in keratinocytes tryptase may be activating a subpopulation of PAR-2 receptors. Treatment of keratinocytes or fibroblasts with human chymase did not produce Ca2+ mobilization, nor did it affect Ca2+ mobilization produced by trypsin. However, chymase pretreatment of fibroblasts rapidly inhibited the ability of these cells to respond to thrombin. Inhibition was dependent on chymase enzymatic activity and was not significantly affected by the presence of heparin. This finding is consistent with studies indicating that PAR-1 may be susceptible to proteases with chymotrypsin-like specificity. These results suggest that the proteases tryptase and chymase secreted from mast cells in skin may affect the behavior of surrounding cells by the hydrolysis of PARs expressed by these cells.
蛋白酶激活受体（PARs）构成了一类通过蛋白水解作用激活的G蛋白信号转导受体家族。在本研究中，评估了人类角质形成细胞和真皮成纤维细胞上表达的PARs对人类肥大细胞蛋白酶类胰蛋白酶和糜蛋白酶的敏感性。通过监测装载荧光Ca2+探针Fura-2的细胞中的胞质[Ca2+]来测量PAR的激活情况。类胰蛋白酶在角质形成细胞中产生瞬时胞质Ca2+动员，但在成纤维细胞中未产生。角质形成细胞中的Ca2+动员需要具有酶活性的类胰蛋白酶，表现出脱敏现象，并且通过用PAR-2肽激动剂SLIGKV、胰蛋白酶或磷脂酶抑制剂U73122预处理细胞而被阻断。肝素是一种与类胰蛋白酶结合并稳定其功能形式的糖胺聚糖，它也抑制类胰蛋白酶诱导的Ca2+动员。类胰蛋白酶引发的最大反应小于用胰蛋白酶（一种已知的PAR-2激活剂）处理角质形成细胞时观察到的反应，并且通过用蛋白酶预处理而对类胰蛋白酶产生不应性的角质形成细胞对胰蛋白酶仍有反应。用凝血酶（PAR-1和-3的激活剂，即凝血酶受体）预处理角质形成细胞对类胰蛋白酶或胰蛋白酶反应没有可检测到的影响。这些数据表明，在角质形成细胞中，类胰蛋白酶可能正在激活PAR-2受体的一个亚群。用人糜蛋白酶处理角质形成细胞或成纤维细胞不会产生Ca2+动员，也不影响胰蛋白酶产生的Ca2+动员。然而，用糜蛋白酶预处理成纤维细胞会迅速抑制这些细胞对凝血酶的反应能力。抑制作用取决于糜蛋白酶的酶活性，并且不受肝素存在的显著影响。这一发现与表明PAR-1可能对具有胰凝乳蛋白酶样特异性的蛋白酶敏感的研究一致。这些结果表明，皮肤中肥大细胞分泌的蛋白酶类胰蛋白酶和糜蛋白酶可能通过水解这些细胞表达的PARs来影响周围细胞的行为。
