Cathepsin D Is the Primary Protease for the Generation of Adenohypophyseal Vasoinhibins: Cleavage Occurs within the Prolactin Secretory Granules

Abstract

Vasoinhibins are a family of N-terminal prolactin (PRL) fragments that inhibit blood vessel growth, dilation, permeability, and survival. The aspartyl endoprotease cathepsin D is active at acidic pH and can cleave rat PRL to generate vasoinhibins. We investigated whether and where vasoinhibins could be generated by cathepsin D in the adenohypophysis of rats and mice and whether their production could be gender dependent. Vasoinhibins were detected in primary cultures of rat adenohypophyseal cells by Western blot with antibodies directed against the N terminus of PRL but not the C terminus. Ovariectomized, estrogen-treated females show greater levels of adenohypophyseal vasoinhibins than males. Peptide sequencing analysis revealed that the cleaved form of PRL in rat adenohypophyseal extracts contains the PRL N terminus and a second N terminus starting at Ser(149), the reported cleavage site of cathepsin D in rat PRL. In addition, cathepsin D inhibition by pepstatin A reduced vasoinhibin levels in rat adenohypophyseal cell cultures. Confocal and electron microscopy showed the colocalization of cathepsin D and PRL within rat adenohypophyseal cells and secretory granules, and a subcellular fraction of rat adenohypophysis enriched in secretory granules contained cathepsin D activity able to generate vasoinhibins from PRL. Of note, vasoinhibins were absent in the adenohypophysis of mice lacking the cathepsin D gene but not in wild-type mice. These findings show that cathepsin D is the main protease responsible for the generation of adenohypophyseal vasoinhibins and that its action can take place within the secretory granules of lactotrophs.

Regulation of Blood Vessels by Prolactin and Vasoinhibins

“…Accordingly, vasoinhibins can be generated by cathepsin D during the pituitary PRL secretory process and thus, subjected to regulated release. Along this line, estradiol increases the synthesis and activity of cathepsin D [24], and the production of anterior pituitary vasoinhibins is higher in females [23], increases at proestrus with respect to diestrus, and estrogens stimulate their release [25].…”

“…Cathepsin D is catalytically active at acidic pH (pH < 5.5), and recent findings showed that it is the main vasoinhibin-generating enzyme in anterior pituitary lactotrophs [23]. Cathepsin D is located in PRL secretory granules, which are acidic, and cathepsin-D null mice are devoid of vasoinhibins in the anterior pituitary gland [23].…”

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

“…Under normal conditions, a pericellular acidic pH can be produced by the activity of the Na C /H C exchanger and H C /ATPase (Piwnica et al 2006). In rat anterior pituitary cells, Cath D was present in PRL secretory granules and has been proposed to be the main protease involved in 16 kDa PRL production (Cruz-Soto et al 2009). Our present data show the cleavage of mPRL to N-terminal mPRL fragment in media from cultures of MG fragments, but this reaction did not occur in the absence of MG fragments, supporting the hypothesis that mature Cath D secreted from MECs produces the N-terminal mPRL fragment in the extracellular milieu.…”

“…Cleavage of rat PRL by Cath D produces a single 16 kDa vasoinhibin (amino acids (aa) 1-145), whereas from human PRL, this enzyme generates vasoinhibins 1-132 (15 kDa), , and 1-150 (17.2 kDa). All these cleavages of full-length native PRL by proteases generated N-terminal 14-18 kDa fragments (Cruz-Soto et al 2009). …”

La prolactine et son fragment 16 kDa dans les tissus de mammifères

“…Des formes clivées, PRL 14 kDa et 16K, ont été détectées dans les granules sécrétoires de l'adénohypophyse [6]. Chez l'homme et plusieurs mammifères, les ARNm et la PRL elle-même sont aussi exprimés dans la glande mammaire et dans le lait, où coexistent diverses formes moléculaires de la PRL (14,18,20,23,25 kDa) [7,8].…”