The atypical PKCs (aPKCs) have been implicated genetically in at least two independent signaling cascades that control NF-kappa B and cell polarity, through the interaction with the adapters p62 and Par-6, respectively. P62 binds TRAF6, which plays an essential role in osteoclastogenesis and bone remodeling. Recently, p62 mutations have been shown to be the cause of the 5q35-linked Paget's disease of bone, a genetic disorder characterized by aberrant osteoclastic activity. Here we show that p62, like TRAF6, is upregulated during RANK-L-induced osteoclastogenesis and that the genetic inactivation of p62 in mice leads to impaired osteoclastogenesis in vitro and in vivo, as well as inhibition of IKK activation and NF-kappa B nuclear translocation. In addition, RANK-L stimulation leads to the inducible formation of a ternary complex involving TRAF6, p62, and the aPKCs. These observations demonstrate that p62 is an important mediator during osteoclastogenesis and induced bone remodeling.

In order to assess the relative roles of the androgenic and/or estrogenic components in the stimulatory effect of dehydroepiandrosterone (DHEA) on bone mineral content (BMC) and density (BMD), ovariectomized (OVX) female rats received DHEA administered alone or in combination with the antiandrogen flutamide (FLU) or the antiestrogen EM-800 for 12 months. We also evaluated, for comparison, the effect of estradiol (E2) and dihydrotestosterone (DHT) constantly released by Silastic implants as well as medroxyprogesterone acetate (MPA) released from poly(lactide-co-glycolide) microspheres. Femoral BMD was decreased by 11% 1 year after OVX, but treatment of OVX animals with DHEA increased BMD to a value 8% above that of intact animals. The administration of FLU reversed by 76% the stimulatory effect of DHEA on femoral BMD and completely prevented the stimulatory effect of DHEA on total body and lumbar spine BMD. Similar results were obtained for BMC. On the other hand, treatment with the antiestrogen EM-800 did not reduce the action of DHEA on BMD or BMC. At the doses used, MPA, E2 and DHT increased femoral BMD, but to a lesser degree than observed with DHEA. Bone histomorphometry measurements were also performed. While DHEA treatment partially reversed the marked inhibitory effect of OVX on the tibial trabecular bone volume, the administration of FLU inhibited by 51% (P < 0.01) the stimulatory effect of DHEA on this parameter. The addition of EM-800 to DHEA, on the other hand, increased trabecular bone volume to a value similar to that of intact controls. DHEA administration markedly increased trabecular number while causing a marked decrease in the intertrabecular area. The above stimulatory effect of DHEA on trabecular number was reversed by 54% (P < 0.01) by the administration of FLU, which also reversed by 29% the decrease in intertrabecular area caused by DHEA administration. On the other hand, the addition of EM-800, while further decreasing the intertrabecular space achieved by DHEA treatment, also led to a further increase in trabecular number to a value not significantly different from that of intact control animals, suggesting an additional effect of EM-800 over that achieved by DHEA. Treatment with DHEA caused a 4-fold stimulation of serum alkaline phosphatase, a marker of bone formation, while the urinary excretion of hydroxyproline, a marker of bone resorption, was decreased by DHEA treatment. Treatment with DHEA and DHEA + EM-800 decreased serum cholesterol levels by 22 and 65% respectively, while the other treatments had no significant effect on this parameter. The present data indicate that the potent stimulatory effect of DHEA on bone in the rat is mainly due to the local formation of androgens in bone cells and their intracrine action in osteoblasts.

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Integrin-mediated cell adhesion and growth factor stimuli are both required for optimal control of cell proliferation. In the context of skin injury, cell-derived fibronectin and platelet-derived growth factor play important roles in the stimulation of cell proliferation and migration, activities that are crucial to the healing process. To assess the ability of exogenously supplied plasma-derived fibronectin to stimulate wound repair and to study its ability to cooperate with platelet-derived growth factor-BB during healing, we devised a novel topical delivery formulation that allows the controlled release of both molecules to a wound. Using this topical formulation and the rabbit ear model of dermal wound healing, we show that plasma fibronectin is a potent stimulator of the wound healing process. We also show that administration of fibronectin and platelet-derived growth factor-BB in combination has additive wound healing effects. Finally, we report novel findings on the ability of soluble plasma fibronectin to potentiate the mitogenic effects of platelet-derived growth factor-BB in vitro. These findings not only show that optimal concentrations of exogenous fibronectin administered using an effective delivery system stimulate wound healing; they also suggest that PDGF-BB should be administered with fibronectin to achieve optimal therapeutic stimulation of wound healing.

Bone destruction in chronic gout is correlated with deposits of monosodium urate (MSU) crystals. Bone with MSU tophi were histopathologically shown to have altered remodeling and cellular distribution. We investigated the impact of neutrophils in bone remodeling associated with MSU and demonstrated that neutrophils, through elastase localized at their surface, induced retraction of confluent osteoblasts (OBs) previously layered on calcified matrix. This OB retraction allowed osteoclasts to resorb cell-free areas of the matrix. This neutrophil effect was concentration dependent and time dependent and required direct contact with OBs. Exposure of OBs to MSU greatly promoted neutrophil adherence to OBs. Neutrophil membrane at the contact zone with OBs showed concentrated fluorescence of dye PKH-67, indicating a cellular contact. Neutrophil-OB interaction increased the survival of neutrophils, reduced their release of lactoferrin in presence of MSU and did not change OB-mediated mineralization. The adhesion of neutrophils to OBs was heterotypic through neutrophil CD29/CD49d and OB-fibronectin peptide CS1. Leukotriene B 4 (LTB 4 ) and platelet-activating factor (PAF) were also involved in neutrophil adherence to OBs, as shown by the blocking effect of selective LTB 4 and PAF receptor antagonists, and a cytosolic phospholipase A 2a (cPLA 2a ) inhibitor. Blockade of CD49d/CS1 and inhibition of the cPLA 2a had subadditive effects, reducing by 60% the adherence of neutrophils to OBs. Taken together, these data showed that neutrophil adhesion to MSU-activated OBs was mediated by the b 1 integrin CD29/CD49d-fibronectin peptide CS1 receptors and cPLA 2a -derived metabolites and impacts on OB and osteoclast functions. These interactions could be involved in the local bone remodeling process of gout.Laboratory Investigation (2011) 91, 905-920;

Sperm surface proteins involved in fertilization can be added or modified during epididymal transit. P34H, a human epididymal-sperm protein, appears on the sperm acrosomal cap in the distal caput-proximal corpus epididymis. In previous studies, it was shown that P34H is present on spermatozoa in men of proven fertility, is absent in 50% of men presenting with idiopathic infertility, and that a high proportion of men with normospermic vasovasectomy produce spermatozoa deficient in this sperm surface protein. P34H mRNA was expressed in the principal cells of the epididymis of normal men, predominantly in the corpus region. Recently, results coming from the assisted reproductive technologies have questioned the importance of the human epididymis in sperm maturation. In order to understand the effect of obstruction on the physiological state of the human epididymis and its function in sperm maturation, we have analyzed the expression of P34H mRNA at the level of the vas deferens and along the epididymis of normal and vasectomized men. In situ hybridization experiments showed that obstruction of the vas deferens alters the pattern of P34H mRNA expression compared with the tract of normal tissues. The P34H transcript was detected in the proximal caput epididymis of vasectomized men at a much higher intensity than that observed in the same region of normal tissues, being restricted to the principal cells of the epididymal epithelium. Compared with the normal duct, the lumen of vasectomized men was distended throughout the duct and the height of the epithelium was maximal in the caput. P34H mRNA was detectable in vas deferens, was not affected by vasectomy, and a 912-base pair P34H transcript was restricted to the epithelial cells of the vas deferens. Thus, using P34H as a marker, these results show that vasectomy alters the pattern of gene expression along the human epididymis, and suggest that the vas deferens can be a major contributor to sperm maturation in certain situations.

The high fatality and morbidity of pancreatic cancer have remained almost unchanged over the last decades and new clinical therapeutic tools are urgently needed. We determined the cytotoxic activity of aminosteroid derivatives RM-133 (androstane) and RM-581 (estrane) in three human pancreatic cancer cell lines (BxPC3, Hs766T and PANC-1). In PANC-1, a similar level of antiproliferative activity was observed for RM-581 and RM-133 (IC = 3.9 and 4.3 μM, respectively), but RM-581 provided a higher selectivity index (SI = 12.8) for cancer cells over normal pancreatic cells than RM-133 (SI = 2.8). We also confirmed that RM-581 induces the same ER stress-apoptosis markers (BIP, CHOP and HERP) than RM-133 in PANC-1 cells, pointing out to a similar mechanism of action. Finally, these relevant in vitro results have been successfully translated in vivo by testing RM-581 using different doses (10-60 mg/kg/day) and modes of administration in PANC-1 xenograft models, which have led to tumor regression without any sign of toxicity in mice (animal weight, behavior and histology). Interestingly, RM-581 fully reduced the pancreatic tumor growth when administered orally in mice.

A link between vascular calcification and bone anomalies has been suggested in chronic kidney disease (CKD) patients with low bone turnover disease. We investigated the vascular expression of osteocyte markers in relation to bone microarchitecture and mineralization defects in a model of low bone turnover CKD rats with vascular calcification. CKD with vascular calcification was induced by 5/6 nephrectomy followed by high calcium and phosphate diet, and vitamin D supplementation (Ca/P/VitD). CKD + Ca/P/VitD group (n = 12) was compared to CKD + normal diet (n = 12), control + normal diet (n = 8) and control + Ca/P/VitD supplementation (n = 8). At week 6, tibia, femurs and the thoracic aorta were analysed by Micro-Ct, histomorphometry and for expression of osteocyte markers. High Ca/P/VitD treatment induced vascular calcification only in CKD rats, suppressed serum parathyroid hormone levels and led to higher sclerostin, DKK1 and FGF23 serum levels. Expression of sclerostin, DKK1 and DMP1 but not FGF23 were increased in calcified vessels from CKD + Ca/P/VitD rats. Despite low parathyroid hormone levels, tibia bone cortical thickness was significantly lower in CKD + Ca/P/VitD rats as compared to control rats fed a normal diet, which is likely the result of radial growth impairment. Finally, Ca/P/VitD treatment in CKD rats induced a bone mineralization defect, which is likely explained by the high calcitriol dose. In conclusion, Ca/P/VitD supplementation in CKD rats induces expression of osteocyte markers in vessels and bone mineralisation anomalies. Further studies should evaluate the mechanisms of high dose calcitriol-induced bone mineralisation defects in CKD.