Notochordal cells (NCs), characterized by their vacuolated morphology and coexpression of cytokeratin and vimentin intermediate filaments (IFs), form the immature nucleus pulposus (NP) of the intervertebral disc. As humans age, NCs give way to mature NP cells, which do not possess a vacuolated morphology and typically only express vimentin IFs. In light of their concomitant loss, we investigated the relationship between cytosolic vacuoles and cytokeratin IFs, specifically those containing cytokeratin-8 proteins, using a human chordoma cell line as a model for NCs. We demonstrate that the chemical disruption of IFs with acrylamide, F-actin with cytochalasin-D, and microtubules with nocodazole all result in a significant (p < 0.001) decrease in vacuolation. However, vacuole loss was the greatest in acrylamide-treated cells. Examination of the individual roles of vimentin and cytokeratin-8 IFs in the existence of vacuoles was accomplished using small interfering RNA-mediated RNA interference to knock down either vimentin or cytokeratin-8 expression. Reduction of cytokeratin-8 expression was associated with a less-vacuolated cell morphology. These data demonstrate that cytokeratin-8 IFs are involved in stabilizing vacuoles and that their diminished expression could play a role in the loss of vacuolation in NCs during aging. A better understanding of the NCs may assist in preservation of this cell type for NP maintenance and regeneration.
Chemically defined serum-free medium has been shown to maintain the mechanical properties of cartilage allografts better than serum supplemented medium during long-term in vitro culture [1]. Little is known about this beneficial mechanism at a cellular level. Intracellular calcium ([Ca2+]i) signaling is one of the earliest responses in chondrocytes under mechanical stimulation [2]. It was recently found that calcium signaling is involved in the regulation of chondrocyte morphology changes and its short-term anabolic and catabolic responses under mechanical stimulation [3]. In this study we hypothesized that the beneficial mechanisms of serum-free culture could be indicated by the spatiotemporal features of [Ca2+]i signaling of chondrocytes in situ. We aimed to: (i) compare the in situ spontaneous [Ca2+]i responses of chondrocytes cultured in medium with and without serum; (ii) investigate the correlation between the [Ca2+]i responses of chondrocytes and the biomechanical properties of cartilage explants.
Sclerostin failed to restore the expression of the anabolic genes, we investigated the effect of Sclerostin on the activation of Wnt non canonical pathways mediated by Ca2+/CaMKII, JNK and PKC proteins. Methods: Primary murine chondrocytes were cultured with or without Wnt3a conditioned media and in the presence or absence of recombinant Sclerostin. Activation of Ca2+/CaMKII, JNK and PKC pathways was analyzed by Western blotting. The role of the activation of JNK and PKC pathways in the chondrocyte phenotype was investigated using SP600125 and Staurosporin inhibitors respectively. Chondrocyte differentiation was investigated by RT-qPCR through the relative expression of type II and X collagen, Sox9, Aggrecan, MMP-3 & 13 and ADAMTS-4&5 genes and by Western blotting through the protein expression of ADAMTS-4 & 5 and type X Collagen Alcian blue staining and spectrophotometric quantification was also used for analyzing the accumulation of highly sulphated GAG.. Results: Wnt3a increased the gene expression of metalloproteinases such as Adamts-4 & 5 MMP3, 13 and type X collagen. This effect was totally abolished in the presence of Sclerostin. In the other hand, Sclerostin restored partially the expression of type II Collagen, Sox9 and Aggrecan inhibited by Wnt3a. Because of the partial effect of Sclerostin on the anabolic genes, we assessed whether Wnt3a activates b-catenin independent pathways such as JNK, PKC and CaMKbII. Wnt3A promoted the phosphorylation of JNK and PKC without affecting CaMKbII phsphorylation. Moreover, Sclerostin inhibited the phosphorylation of JNK but not that of PKC. We further investigated whether Sclerostininduced inhibition of JNK affects chondrocyte function. We found that Wnt3a decreased the accumulation of highly sulphated GAG while Sclerostin failed in rescuing their amount. Interestingly, Sclerostin was able to rescue the accumulation of highly sulphated GAG and the expression of the anabolic genes when JNK pathway is inhibited by SP600125. Conclusions: We here showed that Wnt3a inhibits the expression of the anabolic genes by activating the non-canonical JNK pathways in chondrocytes. Sclerostin reduced the Wnt-induced activation of JNK pathway. These results show that Sclerostin may play a role in the anabolic activity of chondrocytes.
Zoledronic acid (ZA), an FDA approved bisphosphonate (BP) medicine, is widely used for the treatment of osteoclast-related bone loss diseases [1]. Our previous study has found that systemic administration of ZA could dramatically suppress the development of post-traumatic osteoarthritis (PTOA) in the DMM (destabilization of the medial meniscus) mouse model, a model recapitulating the altered joint loading associated with PTOA [2]. This finding is consistent with a few similar studies using different animal models [3]. However, little is known about the cellular and biochemical mechanisms of BP mediated chondro-protection in PTOA pathogenesis. Studies have shown that PTOA often initiates from the apoptosis and altered metabolism of cartilage chondrocytes. In this study, we will investigate the direct effects of ZA on the metabolisms of chondrocytes using long-term in vitro culture of cartilage allografts. As one of the earliest responses of chondrocytes to mechanical stimulation, intracellular calcium ([Ca 2+] i) signaling is the upstream of numerous mechanotransduction pathways [4]. We hypothesize that the chondro-protective mechanisms of ZA could be represented by the characteristics of [Ca 2+] i signaling of in situ chondrocytes. Our specific aims were to: (i) compare the in situ spontaneous [Ca 2+] i responses of chondrocytes cultured in non-ZA and ZA supplemented environments, and (ii) compare the biomechanical properties of cartilage allografts under the two culture conditions.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.