Overproduction of parathyroid hormone-related protein (PTHRP) occurs in a high proportion of primary breast cancers (PBC) and is strongly implicated in their metastatic spread to bone. Although the PTHRP-receptor (PTHRP-R) is often coexpressed with PTHRP in PBC, its role in regulating breast cancer cell proliferation and metastases to bone remains unclear. The aims of this study were to determine the expression of the PTHRP-R in breast cancer bone metastases (BM) and to investigate the effects of PTHRP-R overexpression on breast cancer cell proliferation. PTHRP-R expression occurred in 85% (11 out of 13) of BM compared with 58% (39 out of 67) of PBC. Median expression was higher (Po0.05) in BM compared with PBC. PTHRP increased cAMP accumulation and DNA synthesis in MCF-7 cells stably overexpressing the PTHRP-R (MCF-7 WTR ) but not in MCF-7 VEC control cells. The increase in DNA synthesis was mimicked by the cAMP pathway activator forskolin. The receptor antagonist PTHRP 7 -34 reduced DNA synthesis in MCF-7 WTR cells, but not MCF-7 VEC cells, indicating that receptor overexpression promotes autocrine PTHRP activity. MCF-7 WTR cells showed increased mitogenic responsiveness to fetal calf serum and reduced doubling times. PTHRP induced weak activation of ERK1 and ERK2 and potentiated their activation by serum growth factors. Collectively these results show that the PTHRP-R is frequently expressed in breast cancer BM and indicate that receptor overexpression drives proliferation via autocrine signals that are mediated via cAMP and ERK pathways.

We appreciate the detailed critical analysis of our work by Maioli and Fortino, who have also been investigating the actions of PTHRP in MCF7 cells. The authors make a number of observations and criticisms of our work, to which we are glad to have the opportunity to respond.The first point raised relates to our discussion of some work published by Falzon and Du (2000) regarding intracrine actions of PTHRP. It is true to say we considered that, because their data showing the intracrine effect required overexpression of PTHRP, such a mechanism was unlikely to operate in our system, in which we showed that endogenous PTHRP production was extremely low. We did not, however, question the physiological relevance of the Falzon group's findings and indeed suggested that it would be interesting to test for the presence of intracrine and autocrine activity in cells overexpressing both ligand and receptor.The second point relates to the dose-responsiveness of MCF-7 cells to PTHRP. Maioli and Fortino's results show that, at PTHRP concentrations approximately five-fold higher than the highest dose employed in our study, PTHRP exerts an inhibitory effect on proliferation. While we agree that this is an interesting difference with our work, it represents a difference in experimental conditions employed and not, as implied, a difference in cellular responsiveness to PTHRP. Indeed, since Maoli and Fortino do not show a dose response experiment, it is unclear how their cells would respond to 125 nM PTHRP. On the same point, as stated in our paper, we did not detect any PTHRP-induced Ca 2 þ signal in parental MCF-7 cells treated with 125 nM PTHRP, but did not examine this at the higher ligand concentrations employed by Maioli and Fortino. We suggest, therefore, that there is no basis for their comment about the likely loss of this pathway in our cells. However, in the light of the data shown by Maioli and Fortino, it would clearly be interesting to conduct a more detailed dose -response analysis of the effects of PTHRP on mitogenesis in MCF-7 cells.The third point raised by Maioli and Fortino alludes to our finding of an increased sensitivity to growth factors in cells overexpressing the PTH1R. We were, of course, aware of the vast literature on tyrosine kinase receptor (TKR) transactivation by G protein-coupled receptors and did not claim to be making a novel observation in that respect. We merely report the phenomenon, which we believe is interesting, but do not, at this stage, have substantive experimental evidence concerning its underlying mechanism. We can say however that our unpublished data indicate that the sensitivity observation is not due to TKR transactivation, and appears instead to involve a synergistic interaction of signalling pathways downstream of the two receptors.The final point relates to the effects of forskolin on proliferation; our results indicate a positive effect of forskolin, whereas Maoli and Fortino report a negative effect. As stated in our paper, different concentrations of forkolin that lead to di...