Martine Bisson

The p62 P392L Mutation Linked to Paget’s Disease Induces Activation of Human Osteoclasts

Mutations of the gene encoding p62/SQSTM1 have been described in Pagets 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.

In vitro pro- and antioxidant properties of estrogens.

The pro- and antioxidant properties of estrogens are subject of debate. The apparent discrepancy is largely caused by the chemical heterogeneity in the estrogen family and by their concentration and the environment in which they are found. To gain some insight into this debate, we determined whether estradiol (E(2)), estrone (E(1)), the 2-, 4- and 16alpha-hydroxyestrogens and also the 2- and 4-methoxyestrogens are: (1) good electron-donors; (2) capable of O(2) consumption and DNA strand break induction; (3) capable of inhibiting lipid peroxidation in vitro. E(2), E(1) and 16alpha-hydroxyestrone (16alpha-OHE(1)) were not pro-oxidants and were rather weak antioxidants, while the 2- and 4-hydroxyestrogens demonstrated both properties inducing DNA strand breaks damage as well as inhibiting lipid peroxidation. The 4-hydroxyestrogens consumed O(2) and induced DNA strand breaks to a level approximately 2.5-fold higher than the 2-hydroxyestrogens, but these hydroxyestrogens exhibited similar antioxidant capacity, as measured by inhibition of lipid peroxidation. The 4-methoxyestrogens cannot induce oxidative damage to DNA but can inhibit lipid peroxidation, although being less potent than the 2-methoxyestrogens and the 2- and 4-hydroxyestrogens. The 2-methoxyestrogens were both potent electron donors and inhibitors of lipid peroxidation. Although 2-methoxyestrogens cannot generate superoxide in vitro, they may also be considered pro-oxidants in vivo.

Transforming Growth Factor-β1 (TGF-β1) Induces Human Osteoclast Apoptosis by Up-regulating Bim

Transforming growth factor-β1 (TGF-β1) is the most abundant TGF-β isoform detected in bone and is an important functional modulator of osteoclasts. TGF-β1 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-β receptors. In the absence of survival factors, TGF-β1 (1 ng/ml) induced osteoclast apoptosis. The expression of activated caspase-9, but not that of caspase-8, was increased by TGF-β1 stimulation, and the rate of TGF-β1-induced apoptosis was significantly lower in the presence of a caspase-9 inhibitor. To study further the mechanisms involved in TGF-β1-induced osteoclast apoptosis, we investigated TGF-β1 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-β1 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-β1. Furthermore, the rate of TGF-β1-induced osteoclast apoptosis was lower when Bim expression was suppressed, and inhibiting the Smad pathway abolished Bim up-regulation following TGF-β stimulation. This could correspond to a regulatory mechanism involved in the inhibition of osteoclast activity by TGF-β1.

Activation of matrix metalloproteinase-2 and -9 by 2- and 4-hydroxyestradiol.

Breast cancer patients frequently develop metastases. This process requires the degradation of extracellular matrix proteins which act as a barrier to tumour cell passage. These proteins can be degraded by proteases, mainly the matrix metalloproteinases (MMPs). MMP-2 and -9 which are frequently detected in breast cancer tissues. ProMMPs are released from cancer cells, and their activation is considered to be a crucial step in metastases development. In breast cancer, estrogen metabolism is altered favouring the accumulation of 2- and 4-hydroxyestradiol (2- and 4-OHE(2)). These estradiol metabolites can generate free radicals. Since reactive species are known activators of proMMPs, this study was designed to determine if the free radicals generated by 2- and 4-OHE(2) can activate proMMP-2 and -9. Activation of MMPs by hydroxyestradiol was determined by monitoring the cleavage of a fluorogenic peptide and by zymography analysis. Both estradiol metabolites activated the MMP-2 and -9. 4-OHE(2) was a more potent activator than 2-OHE(2), which reflects its higher capacity to generate free radicals. ProMMPs activation was mainly mediated through O(2)*-, although the free radical HO* also activated the proMMPs but to a lesser extent. ProMMPs activation was not observed with estrogens that cannot generate free radicals, i.e. estradiol, estrone, 2- and 4-methoxyestradiol, and 16alpha-hydroxyestrone. These results demonstrate that 2- and 4-OHE(2) at a concentration as low as 10(-8)M can activate the proMMP-2 and -9 and might play an important role in the invasion of breast cancer cells.

TRPV-5 Mediates a Receptor Activator of NF-κB (RANK) Ligand-induced Increase in Cytosolic Ca2+ in Human Osteoclasts and Down-regulates Bone Resorption

Most of the signaling effectors located downstream of receptor activator of NF-κB (RANK) activation are calcium-sensitive. However, the early signaling events that lead to the mobilization of intracellular calcium in human osteoclasts are still poorly understood. The Ca2+-sensitive fluorescent probe Fura2 was used to detect changes in the intracellular concentration of Ca2+ ([Ca2+]i) in a model of human osteoclasts. Stimulating these cells with receptor activator of NF-κB ligand (RANKL) induced a rapid and significant increase in [Ca2+]i. Adding extracellular Ca2+ chelators, depleting intracellular stores, and the use of a phospholipase C inhibitor all indicated that the Ca2+ was of extracellular origin, suggesting the involvement of a Ca2+ channel. We showed that none of the classical Ca2+ channels (L-, T-, or R-type) were involved in the RANKL-induced Ca2+ spike. However, the effect of high doses of Gd3+ did suggest that TRP family channels were present in human osteoclasts. The TRPV-5 channel was expressed in osteoclasts and was mainly located in the cellular area in contact with the bone surface. Furthermore, the RNA inactivation of TRPV-5 channel completely inhibited the RANKL-induced increase in [Ca2+]i, which was accompanied in the long term by marked activation of bone resorption. Overall, our results show that RANKL induced a significant increase in [Ca2+]i of extracellular origin, probably as a result of the opening of TRPV-5 calcium channels on the surface of human osteoclasts. Our findings suggest that TRPV-5 contributes to maintaining the homeostasis of the human skeleton via a negative feedback loop in RANKL-induced bone resorption.

Bone morphogenetic protein-9 activates Smad and ERK pathways and supports human osteoclast function and survival in vitro.

BMP-9 is a potent osteogenic factor; however, its effects on osteoclasts, the bone-resorbing cells, remain unknown. To determine the effects of BMP-9 on osteoclast formation, activity and survival, we used human cord blood monocytes as osteoclast precursors that form multinucleated osteoclasts in the presence of RANKL and M-CSF in long-term cultures. BMP-9 did not affect osteoclast formation, but adding BMP-9 at the end of the culture period significantly increased bone resorption compared to untreated cultures, and reduced both the rate of apoptosis and caspase-9 activity. BMP-9 also significantly downregulated the expression of pro-apoptotic Bid, but only after RANKL and M-CSF, which are both osteoclast survival factors, had been eliminated from the culture medium. To investigate the mechanisms involved in the effects of BMP-9, we first showed that osteoclasts expressed some BMP receptors, including BMPR-IA, BMPR-IB, ALK1, and BMPR-II. We also found that BMP-9 was able to induce the phosphorylation of Smad-1/5/8 and ERK 1/2 proteins, but did not induce p38 phosphorylation. Finally, knocking down the BMPR-II receptor abrogated the BMP-9-induced ERK-signaling, as well as the increase in bone resorption. In conclusion, these results show for the first time that BMP-9 directly affects human osteoclasts, enhancing bone resorption and protecting osteoclasts against apoptosis. BMP-9 signaling in human osteoclasts involves the canonical Smad-1/5/8 pathway, and the ERK pathway.

Invasiveness of breast cancer cells MDA-MB-231 through extracellular matrix is increased by the estradiol metabolite 4-hydroxyestradiol.

In malignant breast cancer, estrogen metabolism is altered, favoring the accumulation of hydroxyestradiols, which can generate free radicals. These reactive species can activate matrix metalloproteinases (MMPs), which in turn can hydrolyze the proteins of the extracellular matrix (ECM) that act as a barrier to tumor cell passage. The aim of this study was to determine whether reactive oxygen species generated by 4‐hydroxyestradiol (4‐OHE2) can activate MMP‐2 and then enhance the invasiveness of breast cancer cells MDA‐MB‐231 in vitro. Enzymatic assay and gel zymography demonstrated that 4‐OHE2 at a concentration as low as 10−8 M led to the conversion of proMMP‐2 to active MMP‐2. Activation of proMMP‐2 by 4‐OHE2 was inhibited by the Cu,Zn‐SOD supporting the involvement of the free radical superoxide anion (O  2·− ). Using invasion chambers coated with matrigel (artificial ECM), 4‐OHE2 (10−8 M) enhanced the invasiveness of MDA‐MB‐231 breast cancer cells by 3‐fold. The addition of Cu,Zn‐SOD reduced the invasiveness of MDA‐MB‐231 cells by more than 2‐fold, supporting the involvement of O  2·− generated by 4‐OHE2. Addition of an MMP‐2 inhibitor completely inhibited the enhancement of invasiveness induced by 4‐OHE2, which demonstrates the importance of activating MMP‐2 by 4‐OHE2. On the other hand, estradiol, which does not have a catechol structure, did not generate free radicals, and it could not activate proMMP‐2 or enhance the invasiveness of beast cancer cells. Although these data need to be confirmed in an animal model, this study suggests that the accumulation of 4‐OHE2 in breast tumors could enhance the invasiveness of breast cancer cells.

Involvement of kinase PKC-zeta in the p62/p62P392L-driven activation of NF-κB in human osteoclasts

Mutations of the gene encoding sequestosome1 (SQSTM1/p62), clustering in or near the UBA domain, have been described in Pagets 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.

Increased Concentrations of Prostaglandin D2 During Post-Fracture Bone Remodeling

Objective. To test the hypothesis that increased concentrations of prostaglandin D2 (PGD2) correlate with bone remodeling. Studies using isolated bone cells indicate that PGD2 may be implicated in the regulation of bone homeostasis, with a positive influence on bone anabolism. We studied patients with traumatic fractures and age- and sex-matched healthy controls as an in vivo model of increased bone remodeling. Methods. Thirty-five patients with bone fracture and matched controls were recruited. Urine and sera samples were collected. Urinary 11ß-PGF2α, a PGD2 metabolite, and PGE2 metabolites (PGEM), serum lipocalin-type PGD2 synthase (L-PGDS), bone alkaline phosphatase (bone ALP), and crosslinked C-telopeptides of type I collagen (CTX) were measured. Results. At 5–6 weeks post-fracture, 11ß-PGF2α, L-PGDS, bone ALP, and CTX were significantly increased in the fracture patients compared to controls. PGEM levels were not different between groups. Levels of 11ß-PGF2α and bone ALP were positively correlated, suggesting that PGD2 may be implicated in fracture repair. Conclusion. These results support our working hypothesis that PGD2 could be implicated in the control of bone anabolism in humans.

Alternative splicing in osteoclasts and Paget’s disease of bone

BackgroundMutations in the SQSTM1/p62 gene have been reported in Paget’s disease of bone (PDB), but they are not sufficient to induce the pagetic osteoclast (OC) phenotype. We hypothesized that specific RNA isoforms of OC-related genes may contribute to the overactivity of pagetic OCs, along with other genetic predisposing factors.MethodsAlternative splicing (AS) events were studied using a PCR-based screening strategy in OC cultures from 29 patients with PDB and 26 healthy donors (HD), all genotyped for the p62P392L mutation. Primer pairs targeting 5223 characterized AS events were used to analyze relative isoform ratios on pooled cDNA from samples of the four groups (PDB, PDBP392L, HD, HDP392L). Of the 1056 active AS events detected in the screening analysis, 192 were re-analyzed on non-amplified cDNA from each subject of the whole cohort.ResultsThis analysis led to the identification of six AS events significantly associated with PDB, but none with p62P392L. The corresponding genes included LGALS8, RHOT1, CASC4, USP4, TBC1D25, and PIDD. In addition, RHOT1 and LGALS8 genes were upregulated in pagetic OCs, as were CASC4 and RHOT1 genes in the presence of p62P392L. Finally, we showed that the proteins encoded by LGALS8, RHOT1, USP4, TBC1D25, and PIDD were expressed in human OCs.ConclusionThis study allowed the identification of hitherto unknown players in OC biology, and our findings of a differential AS in pagetic OCs may generate new concepts in the pathogenesis of PDB.