Parathyroid hormone (PTH)-related protein (PTHrP) is the main factor responsible for humoral hypercalcemia of malignancy. Both PTH and PTHrP bind to the common type I PTH/PTHrP receptor (PTHR), thereby activating phospholipase C and adenylate cyclase through various G proteins, in bone and renal cells. However, various normal and transformed cell types, including hypercalcemic Walker 256 (W256) tumor cells, do not produce cAMP after PTHrP stimulation. We characterized the PTHrP receptor and the signaling mechanism upon its activation in the latter cells. Scatchard analysis of PTHrP-binding data in W256 tumor cells revealed the presence of high affinity binding sites with an apparent K(d) of 17 nM, and a density of 90 000 sites/cell. In addition, W256 tumor cells immunostained with an anti-PTHR antibody, recognizing its extracellular domain. Furthermore, reverse transcription followed by PCR, using primers amplifying two different regions in the PTHR cDNA corresponding to the N- and C-terminal domains, yielded products from W256 tumor cell RNA which were identical to the corresponding products obtained from rat kidney RNA. Consistent with our previous findings on cAMP production, 1 microM PTHrP(1-34), in contrast to 10 microg/ml cholera toxin or 1 microM isoproterenol, failed to affect protein kinase A activity in W256 tumor cells. However, in these cells we found a functional PTHR coupling to G(alpha)(q/11), whose presence was demonstrated in these tumor cell membranes by Western blot analysis. Our findings indicate that W256 tumor cells express the PTHR, which seems to be coupled to G(alpha)(q/11). Taken together with previous data, these results support the hypothesis that a switch from the cAMP pathway to the phospholipase C-intracellular calcium pathway, associated with PTHR activation, occurs in malignant cells.
P Esbrit, J Benitez-Verguizas, F de Miguel, A Valin and A Garcia-Ocana
ML Villanueva-Penacarrillo, J Cancelas, F de Miguel, A Redondo, A Valin, I Valverde and P Esbrit
Parathyroid hormone (PTH)-related protein (PTHrP) is present in the pancreatic islet. Recent data in transgenic mice suggest that PTHrP might modulate islet mass and insulin secretion. In the present study, we assessed the effect of the N-terminal PTH-like region of PTHrP on DNA synthesis in isolated rat islets. PTHrP (1-34), between 1 pM and 10 nM, for 48 h stimulated thymidine incorporation into rat islets. This effect was maximally induced, about 2.5-fold over control, by 10 pM of this peptide, decreasing thereafter. In contrast, PTHrP (38-64) amide or PTHrP (107-139) were ineffective in increasing DNA synthesis in islets. Using reverse transcription followed by PCR, we confirmed that rat islets express PTHrP and the type I PTH/PTHrP receptor. Addition of a neutralizing anti-PTHrP antibody to the incubation medium of proliferating islets decreased islet DNA synthesis by 30%. The effect of a submaximal dose (30 pM) of PTHrP (1-34) on DNA synthesis in rat islets was abolished by 25 nM bisindolylmaleimide I, a protein kinase C (PKC) inhibitor, but not by 25 microM adenosine 3',5'-cyclic monophosphorothioate, Rp-isomer, a protein kinase A inhibitor. Moreover, 100 nM phorbol-12-myristate-13-acetate for 48 h also increased DNA synthesis 2-fold over controls in islets. PTHrP (1-34), at 100 nM, in contrast to 50 microM forskolin or 10 mM NaF, failed to affect adenylate cyclase activity in islet membranes. PTHrP, at 30 pM, was also found to increase 2-fold insulin released into the islet-conditioned medium within 24-48 h. Our results suggest that PTHrP is a modulator of pancreatic islet growth and/or function by a PKC-mediated mechanism.