Active Plasma Kallikrein Localizes to Mast Cells and Regulates Epithelial Cell Apoptosis, Adipocyte Differentiation, and Stromal Remodeling during Mammary Gland Involution

The plasminogen cascade of serine proteases directs both development and tumorigenesis in the mammary gland. Plasminogen can be activated to plasmin by urokinase-type plasminogen activator (uPA), tissue-type plasminogen activator (tPA), and plasma kallikrein (PKal). The dominant plasminogen activator for mammary involution is PKal, a serine protease that participates in the contact activation system of blood coagulation. We observed that the prekallikrein gene (Klkb1) is expressed highly in the mammary gland during stromal remodeling periods including puberty and postlactational involution. We used a variant of ecotin (ecotin-PKal), a macromolecular inhibitor of serine proteases engineered to be highly specific for active PKal, to demonstrate that inhibition of PKal with ecotin-PKal delays alveolar apoptosis, adipocyte replenishment, and stromal remodeling in the involuting mammary gland, producing a phenotype resembling that resulting from plasminogen deficiency. Using biotinylated ecotin-PKal, we localized active PKal to connective tissue-type mast cells in the mammary gland. Taken together, these results implicate PKal as an effector of the plasminogen cascade during mammary development.The plasminogen cascade of serine proteases regulates both development and tumorigenesis in the mammary gland (1, 2). The ultimate effector in this cascade, plasminogen as its active form, plasmin, is mediated by an intricate cascade of plasminogen activators and protease inhibitors. Plasminogen-deficient mice exhibit significant defects in lactational competence and post-lactational mammary gland involution (2), the process by which the differentiated, lactating gland remodels after the cessation of lactation to a state approaching that of the non-pregnant animal. The effect of plasminogen loss is exacerbated after a round of pregnancy and lactation: plasminogen-null mammary glands have poorly developed secretory alveoli during lactation, and upon involution, never fully involute. Instead, the secretory alveoli fail to regress normally. Moreover, the stroma becomes fibrotic and is cleared incompletely of partially degraded epithelial basement membrane. Because plasminogen-deficient mice largely are unable to support a second round of pregnancy and lactation (2), this suggests that the involution defect is not overcome by activities of other proteases eventually. These studies establish plasminogen as a crucial protease in normal mammary gland biology.Plasminogen is synthesized in the liver and circulates as a zymogen through blood plasma to all vascularized tissues of the body. As this expression and circulation are constant, activation of the plasminogen cascade must be controlled locally to avoid rampant tissue proteolysis. Accordingly, plasminogen can be activated to plasmin by urokinase-type plasminogen activator (uPA),² tissue-type plasminogen activator (tPA), and plasma kallikrein (3). Though tPA and uPA are efficient and well characterized plasminogen activators, studies of mice singly as well as doubly targeted for deficiency of these plasminogen activators show they do not recapitulate the mammary gland phenotype of plasminogen deficiency (4). Instead, through use of variants of ecotin, a macromolecular inhibitor for serine proteases derived from Escherichia coli, we have previously suggested that the dominant plasminogen activator for mammary stromal involution is plasma kallikrein (PKal) (4).PKal, the activated form of the zymogen prekallikrein encoded by the Klkb1 gene, is an 80-kDa serine protease that also is synthesized in the liver and circulates in plasma at about 40-50 μg/ml. PKal participates in the contact activation system of intrinsic coagulation by activating high molecular weight kininogen into bradykinin (5-8). While plasma kallikrein is so-named due to its bradykinin-generating ability, it is in fact structurally and catalytically distinct from the large family of tissue kallikreins, which activate an alternate form of bradykinin from both high and low molecular weight kininogen (9). Moreover, PKal activates plasminogen into plasmin in vitro (3), albeit less efficiently than uPA and tPA.To determine the role of PKal in plasminogen activation in vivo in mammary gland involution, we used a variant of ecotin that was engineered to be highly specific for active PKal (10). This ecotin variant, named ecotin-PKal, inhibits plasminogen activation in vivo in a model of wound healing (11). In this study, we demonstrate that inhibition of PKal significantly delays mammary gland involution.