We recently reported that the ubiquitous, secreted protein clusterin has chaperone activity in vitro [Humphreys et al. (1999) J. Biol. Chem. 274, 6875-6881]. In this study, we demonstrate that clusterin (i) inhibits stress-induced precipitation of a very broad range of structurally divergent protein substrates, (ii) binds irreversibly via an ATP-independent mechanism to stressed proteins to form solubilized high molecular weight complexes, (iii) lacks detectable ATPase activity, (iv) when acting alone, does not effect refolding of stressed proteins in vitro, and (v) stabilizes stressed proteins in a state competent for refolding by heat shock protein 70 (HSP70). Furthermore, we show that, at physiological levels, clusterin inhibits stress-induced precipitation of proteins in undiluted human serum. Clusterin represents the first identified secreted mammalian chaperone. However, reports from others suggest that, at least under stress conditions, clusterin may be retained within cells to exert a protective effect. Regardless of the topological site(s) of action, the demonstration that clusterin can stabilize stressed proteins in a refolding-competent state suggests that, during stresses, the action of clusterin may inhibit rapid and irreversible protein precipitation and produce a reservoir of inactive but stabilized molecules from which other refolding chaperones can subsequently salvage functional proteins.

Background and purpose: Strontium ranelate reduces fracture risk in postmenopausal women with osteoporosis. Evidence from non-clinical studies and analyses of bone markers in phase III trials indicate that this is due to an increase in osteoblast formation and a decrease of osteoclastic resorption. The aim of this work was to investigate, in human cells, the mechanisms by which strontium ranelate is able to influence the activities of osteoblasts and osteoclasts. Experimental approach: Human primary osteoblasts were used to examine effects of strontium ranelate on replication (thymidine incorporation), differentiation (Runx2 and alkaline phosphatase) and cell survival (cell counts and caspase activity). Osteoprotegerin (OPG) was measured by quantitative reverse transcription PCR (qRT-PCR) and ELISA and receptor activator of NFkB ligand (RANKL) by qRT-PCR and Western blot. As strontium ranelate has been proposed as an agonist of the calciumsensing receptor (CaSR), the involvement of CaSR in the effects of strontium ranelate on OPG and RANKL expression, and cell replication was examined using siRNA. Key results: Strontium ranelate increased mRNA and protein levels of OPG and suppressed those of RANKL. Strontium ranelate also stimulated osteoblast replication and differentiation and increased cell survival under stress. Knocking down CaSR suppressed strontium ranelate-induced stimulation of OPG mRNA, reduction of RANKL mRNA, and increase in replication, indicating the involvement of CaSR in these responses. Conclusions and implications:Our results demonstrate that osteoblasts play a key role in the mechanism of action of the anti-fracture agent, strontium ranelate by mediating both its anabolic and anti-resorptive actions, at least in part, via activation of CaSR.

Exposure to ultraviolet radiation (UVR) can lead to a range of deleterious responses in the skin. An important form of damage is the DNA photolesion cyclobutane pyrimidine dimer (CPD). CPDs can be highly mutagenic if not repaired prior to cell division and can lead to UV-induced immunosuppression, making them potentially carcinogenic. UVR exposure also produces vitamin D, a prehormone. Different shapes of the steroid hormone 1a,25-dihydroxyvitamin D 3 [1,25(OH) 2 D 3 ] can produce biological responses through binding either to its cognate nuclear receptor (VDR) to regulate gene transcription or to the VDR associated with plasma membrane caveolae to produce, via signal transduction, nongenomic physiologic responses. Here, we show that both 1,25(OH) 2 D 3 and 1a,25(OH) 2 -lumisterol (JN), a conformationally restricted analogue that can generate only nongenomic responses, are effective inhibitors of UV damage in an immunocompetent mouse (Skh:hr1) model susceptible to UV-induced tumors. Both 1,25(OH) 2 D 3 and JN significantly reduced UVR-induced CPD, apoptotic sunburn cells, and immunosuppression. Furthermore, these compounds inhibited skin tumor development, both papillomas and squamous cell carcinomas, in these mice. The observed reduction of these UV-induced effects by 1,25 (OH) 2 D 3 and JN suggests a role for these compounds in prevention against skin carcinogenesis. To the best of our knowledge, this is the first comprehensive report of an in vivo long-term biological response generated by chronic dosing with a nongenomic-selective vitamin D steroid.