Mutations of the gene encoding p62/SQSTM1 have been described in Paget's disease of bone (PDB), identifying p62 as an important player in osteoclast signaling. We investigated the phenotype of osteoclasts differentiated from peripheral blood monocytes obtained from healthy donors or PDB patients, all genotyped for the presence of a mutation in the p62 ubiquitin-associated domain. The cohort included PDB patients carrying or not the p62 P392L mutation and healthy donors carrying or not this mutation. Osteoclasts from PDB patients were more numerous, contained more nuclei, were more resistant to apoptosis, and had a greater ability to resorb bone than their normal counterparts, regardless of whether the p62 mutation was present or not. A strong increase in p62 expression was observed in PDB osteoclasts. The presence of the p62(P392L) gene in cells from healthy carriers conferred a unique, intermediate osteoclast phenotype. In addition, we report that two survival-promoting kinases, protein kinase Czeta and phosphoinositide-dependent protein kinase 1, were associated with p62 in response to receptor activator of NF-kappaB ligand (RANKL) stimulation in controls and before RANKL was added in PDB osteoclasts. In transfected osteoclasts derived from cord blood monocytes, the p62 P392L mutation contributed to increased activation of kinases protein kinase Czeta/lambda and phosphoinositide-dependent protein kinase 1, along with basal activation of NF-kappaB, independently of RANKL stimulation. These findings clearly indicate that the overexpression of p62 in PDB patients induces important shifts in the pathways activated by RANKL and up-regulates osteoclast functions. Moreover, the most-commonly reported p62 mutation, P392L, certainly contributes to the overactive state of osteoclasts in PDB.

Transforming growth factor-␤1 (TGF-␤1) is the most abundant TGF-␤ isoform detected in bone and is an important functional modulator of osteoclasts. TGF-␤1 can induce osteoclast apoptosis; however, the apoptotic pathways involved in this process are not known. We show here that human osteoclasts express both type-I and type-II TGF-␤ receptors. In the absence of survival factors, TGF-␤1 (1 ng/ml) induced osteoclast apoptosis. The expression of activated caspase-9, but not that of caspase-8, was increased by TGF-␤1 stimulation, and the rate of TGF-␤1-induced apoptosis was significantly lower in the presence of a caspase-9 inhibitor. To study further the mechanisms involved in TGF-␤1-induced osteoclast apoptosis, we investigated TGF-␤1 signaling, which primarily involves the Smad pathway, but also other pathways that may interfere with intracellular modulators of apoptosis, such as mitogen-activated protein (MAP) kinases and Bcl2 family members. We show here that early events consisted of a trend toward increased expression of extracellular signal-regulated kinase (ERK), and then TGF-␤1 significantly induced the activation of p38 and Smad2 in a time-dependent manner. These signaling cascades may activate the intrinsic apoptosis pathway, which involves Bim, the expression of which was increased in the presence of TGF-␤1. Furthermore, the rate of TGF-␤1-induced osteoclast apoptosis was lower when Bim expression was suppressed, and inhibiting the Smad pathway abolished Bim up-regulation following TGF-␤ stimulation. This could correspond to a regulatory mechanism involved in the inhibition of osteoclast activity by TGF-␤1.

In their letter, Labrinidis and colleagues raise the important issue of what (if any) might be the role of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in osteoclastogenesis. In previous studies, we 1,2 and 2 other independent groups of investigators 3-5 have shown that histidine-tagged (His-tag) recombinant TRAIL negatively regulates osteoclastogenesis by inhibiting preosteoclast differentiation and by inducing apoptosis of mature osteoclasts. On the other hand, Labrinidis et al were unable to confirm these previous findings when exposing osteoclastic cultures to the version of Apo2L/TRAIL that is currently being used in phase 1b clinical trials. Although Labrinidis et al emphasize the differences between the recombinant TRAIL preparations used in their and previous studies, 1-5 the possibility that the antiosteoclastic activity of TRAIL merely reflects an aspecific toxic effect of recombinant His-tag TRAIL is ruled out by 2 major considerations: (1) different groups of investigators have clearly documented the ability of recombinant His-TRAIL to induce in vitro prosurvival and even proliferative responses in a cell-type specific manner 6 ; and (2) Roux's group has recently demonstrated that native TRAIL, produced and released in vitro by end-stage osteoclasts, promotes osteoclastic apoptosis through autocrine/paracrine mechanism. 4 Thus, besides blocking receptor-activator of NF-B ligand (RANKL)-mediated osteoclastogenesis, osteoprotegerin (OPG) seems also able to protect mature osteoclasts from apoptosis mediated by native TRAIL endogenously produced by osteoclasts. 4 These findings corroborate the hypothesis that the relative concentrations of RANKL, OPG, and TRAIL at the local bone marrow level are critical for determining the fate of osteoclasts. 6,7 The net effect of TRAIL on osteoclastic differentiation and survival likely depends on the network of prosurvival and proapoptotic signals operating at a given time in the bone marrow microenvironment. In this respect, it should be considered that the antiosteoclastic activity of TRAIL reported by our and other groups 1-5 was observed in culture conditions in which purified populations of preoste-oclasts were induced to differentiate along the osteoclastic lineage by adding recombinant macrophage-colony stimulating factor (M-CSF) plus RANKL to the culture medium. On the other hand, Labrinidis et al have cultured peripheral blood mononuclear cells (used as a source of preosteoclasts) in the presence also of vitamin D3 and dexamethasone, which are known to potently promote osteclastic survival and differentiation. 8 Thus, in our view, the novel contribution of the findings of Labrinidis et al with respect to previous data 1-5 relies on the demonstration that the presence in culture of vitamin D3 and dexamethasone abrogates the antidiffer-entiative and proapoptotic activities of TRAIL. However, this does not exclude a role of TRAIL in osteoclastogenesis, as suspected by Labrinidis et al, but rather suggests a level of molecular control on the a...

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The specific development of the human fetal adrenal gland requires cell proliferation, migration, apoptosis, and zone-specific steroidogenic activity. The present work was designed to determine the physiological significance of the previously identified spatial distribution of extracellular matrix components in the fetal gland. Primary cultures of human fetal adrenal cells grown on collagen IV, laminin, or fibronectin revealed that cell morphology was affected by environmental cues. Matrices also modulated the profile of steroid secretion by the fetal cells. Collagen IV favored cortisol secretion after ACTH or angiotensin II stimulation and increased dehydroepiandrosterone production when the AT(2) receptor of angiotensin II was specifically stimulated. These effects were correlated by changes in the mRNA levels of 3beta-hydroxysteroid dehydrogenase and cytochrome P450C17. In contrast, fibronectin and laminin decreased cell responsiveness to ACTH in terms of cortisol secretion, but enhanced ACTH-stimulated androgen secretion. Finally, extracellular matrices were able to orchestrate cell behavior. Collagen IV and laminin enhanced cell proliferation, and fibronectin increased cell death. This study is the first to demonstrate that the nature of extracellular matrix coordinates specific steroidogenic pathways and cell turnover in the developing human fetal adrenal gland.

The development of the human fetal adrenal gland is characterized by a gradient of mitotic activity, cell migration, and cell apoptosis, all of which dictate its particular function. Such plasticity may possibly be under the control of the extracellular environment. The goal of this study was to identify components of the extracellular matrix in second-trimester fetal adrenal glands. Whereas collagen IV was expressed evenly throughout the gland, both fibronectin and laminin demonstrated a mirror-imaged distribution, with higher expression of fibronectin in the central portion and laminin at the periphery of the gland. The integrin subunit alpha1 was found mainly in the definitive zone and the alpha2-subunit mainly in the transitional zone, whereas integrin alpha3 (which binds both fibronectin and laminin) was detected only in the fetal zone. The beta2-subunit was observed solely in chromaffin cells. Such specific gradients of integrin and MEC component expression suggest that the extracellular environment does play a definite role during adrenal gland development. Indeed, compared with that in untreated plastic dishes, ACTH stimulation of dehydroepiandrosterone sulfate and cortisol was enhanced by collagen IV. In addition, fibronectin enhanced dehydroepiandrosterone sulfate but decreased cortisol secretion, compared with collagen IV substrates. These results provide fundamental insight into the contribution of the microenvironment in cellular processes leading to fetal adrenal gland development.

Mutations of the gene encoding sequestosome1 (SQSTM1/p62), clustering in or near the UBA domain, have been described in Paget's disease of bone (PDB); among these the P392L substitution is the most prevalent. Protein p62 mediates several cell functions, including the control of NF-κB signaling, and autophagy. This scaffolding protein interacts with atypical PKCζ in the RANKL-induced signaling complex. We have previously shown that osteoclasts (OCs) overexpressing the p62(P392L) variant were in a constitutively activated state, presenting activated kinase p-PKCζ/λ and activated NF-κB prior to RANKL stimulation. In the present study, we investigated the relationships between PKCζ and NF-κB activation in human OCs transfected with p62 variants. We showed that PKCζ and p-PKCζ/λ co-localize with p62, and that PKCζ is involved in the RANKL-induced NF-κB activation and in the RANKL-independent activation of NF-κB observed in p62(P392L)-transfected cells. We also observed a basal and RANKL-induced increase in IκBα levels in the presence of the p62(P392L) mutation that contrasted with the NF-κB activation. In this study we propose that PKCζ plays a role in the activation of NF-κB by acting as a p65 (RelA) kinase at Ser(536), independently of IκBα; this alternative pathway could be used preferentially in the presence of the p62(P392L) mutation, which may hinder the ubiquitin-proteasome pathway. Overall, our results highlight the importance of p62-associated PKCζ in the overactive state of pagetic OCs and in the activation of NF-κB, particularly in the presence of the p62(P392L) mutation.

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