Opposing mitogenic and anti-mitogenic actions of parathyroid hormone-related protein in vascular smooth muscle cells: A critical role for nuclear targeting (cellular proliferationyalternative translational start siteynucleolus)

Parathyroid hormone-related protein (PTHrP) is a prohormone that is posttranslationally processed to a family of mature secretory forms, each of which has its own cognate receptor(s) on the cell surface that mediate the actions of PTHrP. In addition to being secreted via the classical secretory pathway and interacting with cell surface receptors in a paracrineyautocrine fashion, PTHrP appears to be able to enter the nucleus directly following translation and influence cellular events in an ‘‘intracrine’’ fashion. In this report, we demonstrate that PTHrP can be targeted to the nucleus in vascular smooth muscle cells, that this nuclear targeting is associated with a striking increase in mitogenesis, that this nuclear effect on proliferation is the diametric opposite of the effects of PTHrP resulting from interaction with cell surface receptors on vascular smooth muscle cells, and that the regions of the PTHrP sequence responsible for this nuclear targeting represent a classical bipartite nuclear localization signal. This report describes the activation of the cell cycle in association with nuclear localization of PTHrP in any cell type. These findings have important implications for the normal physiology of PTHrP in the many tissues which produce it, and suggest that gene delivery of PTHrP or modified variants may be useful in the management of atherosclerotic vascular disease. Parathyroid hormone-related protein (PTHrP) (Fig. 1), isolated in 1987, is responsible for the common endocrine paraneoplastic syndrome, humoral hypercalcemia of malignancy (1). PTHrP is now known to be produced by most cells and tissues in the body (1–4). Following translation, PTHrP enters the secretory pathway and, in cell types that posses the regulated secretory pathway such as pancreatic islet cells and atrial cardiocytes, it is packaged into secretory granules and is subject to regulated secretion (5, 6). In tissues that lack the regulated secretory pathway, such as squamous carcinoma cells and fibroblasts, it is secreted constitutively (5, 6). During its transit through the secretory pathway, the precursor is endoproteolytically processed at basic residues to yield a family of mature secretory forms of the peptide (Fig. 1) (2, 3, 7). Other secretory forms are certain to exist, including peptides with the approximate compositions of PTHrP(107–139) and PTHrP(141–173). Each of the secretory forms of PTHrP appears to act via cell surface receptors (8–12). In addition to its secretion via the classical secretory pathway, other researchers have provided evidence that PTHrP may signal via translocation to the nucleus or nucleolus (13–15). Recognizing that the multibasic clusters in the 88–106 region are similar to nuclear or nucleolar localization signals in viral and mammalian transcription factors (13–16), these reseachers demonstrated that PTHrP can be observed in the nucleolus of chondrocytes and transfected COS cells, that deletion of the multibasic 87–106 region prevents this targeting, and that expression of this multibasic 87–106 region as a fusion protein with b-galactosidase directs this latter protein to the nucleolus (15). In addition, Henderson et al. (14) have suggested that nuclear targeting of PTHrP inhibits apoptosis in chondrocytes. PTHrP is expressed in arterial smooth muscle and is upregulated by mechanical stretch, by hypertension, by vasoconstrictors such as angiotensin II, and by mechanical injury such as angioplasty (17). It is expressed at higher than normal levels in atherosclerotic human coronary arteries (18). PTHrP has been shown to be a potent vasodilator and hypotensive agent (17). Pirola et al. (19), Maeda et al. (20), and Jiang et al. (21) have all demonstrated that PTHrP is an inhibitor of vascular smooth muscle (VSM) cell proliferation, and that PTHrP acts in smooth muscle via the cell surface PTHyPTHrP receptor. We were interested in studying the posttranslational processing of PTHrP in VSM cells, and therefore stably transfected a commonly used rat smooth muscle cell line, A-10, with a PTHrPencoding plasmid. To our surprise, and in contrast to the inhibition of proliferation observed by other researchers (19–21), stable transfection with PTHrP dramatically stimulated VSM cell proliferation. These observations have important mechanistic implications for the actions of PTHrP in the several transgenic mouse models of targeted PTHrP overexpression and for the physiologic actions of PTHrP in the myriad tissues that express the peptide under normal conditions.