Vasoinhibins, N-terminal mouse prolactin fragments, participate in mammary gland involution

Abstract

Vasoinhibins are a family of peptides that act on endothelial cells to suppress angiogenesis and promote apoptosis-mediated vascular regression. Vasoinhibins include the N-terminal fragments from prolactin (PRL), GH, and placental lactogen. One of the vasoinhibins, the N-terminal PRL fragment of 16 kDa, is generated by the lysosomal representative protease cathepsin D (Cath D). Because the normal growth and involution of the mammary gland (MG) are profoundly affected by the expansion and regression of blood vessels and also because PRL stimulates the growth and differentiation of MG, we proposed that intact PRL produced during lactation contributes to MG angiogenesis and increased blood flow, whereas during involution, the N-terminal PRL fragment would have proapoptotic effects on mammary epithelial cells (MECs). Therefore, we investigated the production of the N-terminal PRL fragment and its direct effect on the MG. Mouse PRL (mPRL) was proteolytically cleaved by Cath D between amino acids 148 and 149. N-terminal PRL fragment and Cath D expression increased during MG involution. Furthermore, incubation of MG fragments and MCF7 with recombinant 16 kDa mPRL revealed a proapoptotic effect in MECs. Ectopic mPRL in MECs was cleaved to 16 kDa PRL by Cath D in the MG lysosomal fraction. The majority of PRL derived from pituitary gland was cleaved to 16 kDa PRL in culture medium. Therefore, N-terminal PRL fragment increases during the involution period, has a proapoptotic effect on MECs, and is mainly generated by secreted Cath D in the extracellular space of MG.

Regulation of Blood Vessels by Prolactin and Vasoinhibins

“…Consistent with the ubiquitous nature of PRL-cleaving enzymes, endogenous vasoinhibins have been identified in the anterior [23] and posterior pituitary gland [31], hypothalamus [32], cartilage [20], retina [33], cardiomyocytes [13], corpus luteum [17], mammary gland [16], and in biological fluids (serum, amniotic fluid, and urine) [34,35].…”

“…4.1). Cathepsin D cleaves rat PRL and mouse PRL into a 16 kDa N-terminal fragment [15,16], bovine PRL into 14 and 16 kDa N-terminal fragments [17], buffalo PRL into 11, 14, and 18 kDa antiangiogenic fragments [18], and human PRL into 11, 15, 16.5, and 17 kDa N-terminal fragments [19]. Matrix metalloproteases (MMP) predominantly cleave human and rat PRL at amino acids 155 and 153, respectively, to generate 17 kDa N-terminal fragments [20], and bone morphogenetic protein-1 (BMP-1) cleaves after the first 159 amino acids of human and mouse PRL, generating an 18 kDa fragment [21].…”

“…Cathepsin D cleaves PRL in the extracellular milieu of regressing corpus luteum [17] and mammary gland [16], and in cardiomyocytes under oxidative stress [13], conditions in which tissue remodelling and altered metabolic activity can acidify the pericellular pH. Cultured GH4C1 pituitary adenoma cells also secrete cathepsin D, and mimicking the tumor microenvironment by exposure to hypoxia reduces its release [26], suggesting that extracellular production of vasoinhibins could be decreased and favor the proangiogenic condition necessary for prolactinoma growth.…”

“…PRL promotes the expression of VEGF in mammary epithelial cells [36], and weaning upregulates the expression and activity of the vasoinhibin-generating proteases, MMP, and cathepsin D [59]. Vasoinhibins were recently detected in mouse mammary glands, and their levels increased during involution together with those of the mature cathepsin D isoform [16]. Moreover, PRL stimulates the activation and polarized secretion of cathepsin D by mammary tissue [60].…”

“…Moreover, PRL stimulates the activation and polarized secretion of cathepsin D by mammary tissue [60]. This mechanism may help attenuate vascular expansion during lactation and promote blood vessel regression during involution since PRL expression and cathepsin D-mediated PRL cleavage increase in the lactating and regressing mammary gland [16,36]. However, the mechanisms regulating the antiangiogenic effects of vasoinhibins may be altered in the malignant state.…”