Sphingosine-1-Phosphate Modulates the Effect of Estrogen in Human Osteoblasts

Production of sphingosine‐1‐phosphate (S1P) is linked to 17β‐estradiol (E2) activity in many estrogen‐responsive cells; in bone development, the role of S1P is unclear. We studied effects of S1P on proliferation and differentiation of human osteoblasts (hOB). Ten nM E2, 1 μM S1P, or 1 μM of the S1P receptor 1 (S1PR1) agonist SEW2871 increased hOB proliferation at 24 hours. S1PR 1, 2, and 3 mRNAs are expressed by hOB but not S1PR4 or S1PR5. Expression of S1PR2 was increased at 7 and 14 days of differentiation, in correspondence with osteoblast‐related mRNAs. Expression of S1PR1 was increased by E2 or S1P in proliferating hOB, whereas S1PR2 mRNA was unaffected in proliferating cells; S1PR3 was not affected by E2 or S1P. Inhibiting sphingosine kinase (SPHK) activity with sphingosine kinase inhibitor (Ski) greatly reduced the E2 proliferative effect. Both E2 and S1P increased SPHK mRNA at 24 hours in hOB. S1P promoted osteoblast proliferation via activating MAP kinase activity. Either E2 or S1P increased S1P synthesis in a fluorescent S1P assay. Interaction of E2 and S1P signaling was indicated by upregulation of E2 receptor mRNA after S1P treatment. E2 and S1P also promoted alkaline phosphatase expression. During osteoblast differentiation, S1P increased bone‐specific mRNAs, similarly to the effects of E2. However, E2 and S1P showed differences in the activation of some osteoblast pathways. Pathway analysis by gene expression arrays was consistent with regulation of pathways of osteoblast differentiation; collagen and cell adhesion proteins centered on Rho/Rac small GTPase signaling and Map kinase or signal transducer and activator of transcription (Stat) intermediates. Transcriptional activation also included significant increases in superoxide dismutase 1 and 2 transcription by either S1P or E2. We demonstrate that the SPHK system is a co‐mediator for osteoblast proliferation and differentiation, which is mainly, but not entirely, complementary to E2, whose effects are mediated by S1PR1 and S1PR2. © 2018 The Authors JBMR Plus is published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research

Chemomodulation of human dendritic cell function by antineoplastic agents in low noncytotoxic concentrations

The dose-delivery schedule of conventional chemotherapy, which determines its efficacy and toxicity, is based on the maximum tolerated dose. This strategy has lead to cure and disease control in a significant number of patients but is associated with significant short-term and long-term toxicity. Recent data demonstrate that moderately low-dose chemotherapy may be efficiently combined with immunotherapy, particularly with dendritic cell (DC) vaccines, to improve the overall therapeutic efficacy. However, the direct effects of low and ultra-low concentrations on DCs are still unknown. Here we characterized the effects of low noncytotoxic concentrations of different classes of chemotherapeutic agents on human DCs in vitro. DCs treated with antimicrotubule agents vincristine, vinblastine, and paclitaxel or with antimetabolites 5-aza-2-deoxycytidine and methotrexate, showed increased expression of CD83 and CD40 molecules. Expression of CD80 on DCs was also stimulated by vinblastine, paclitaxel, azacytidine, methotrexate, and mitomycin C used in low nontoxic concentrations. Furthermore, 5-aza-2-deoxycytidine, methotrexate, and mitomycin C increased the ability of human DCs to stimulate proliferation of allogeneic T lymphocytes. Thus, our data demonstrate for the first time that in low noncytotoxic concentrations chemotherapeutic agents do not induce apoptosis of DCs, but directly enhance DC maturation and function. This suggests that modulation of human DCs by noncytotoxic concentrations of antineoplastic drugs, i.e. chemomodulation, might represent a novel approach for up-regulation of functional activity of resident DCs in the tumor microenvironment or improving the efficacy of DCs prepared ex vivo for subsequent vaccinations

Creatine energy substrate increases bone density in the Pahenu2 classical PKU mouse in the context of phenylalanine restriction

Pathophysiology of osteopenia in phenylalanine hydroxylase (PAH) deficient phenylketonuria (PKU) is poorly characterized. The Pahenu2 mouse is universally osteopenic where dietary phenylalanine (Phe) management with amino acid defined chow does not improve bone density. We previously demonstrated Pahenu2 osteopenia owes to a skeletal stem cell (SSC) developmental deficit mediated by energy dysregulation and oxidative stress. This investigation demonstrates complexity of Pahenu2 SSC energy dysregulation. Creatine use by bone tissue is recognized. In vitro Pahenu2 SSCs in osteoblast differentiation respond to creatine with increased in situ alkaline phosphatase activity and increased intracellular ATP content. Animal studies applied a 60-day creatine regimen to Pahenu2 and control cohorts. Control cohorts include unaffected littermates (wt/wt), Pahenu2 receiving no intervention, and dietary Phe restricted Pahenu2. Experimental cohorts (Phe unrestricted Pahenu2, Phe restricted Pahenu2) were provided 1% creatine ad libitum in water. After 60 days, microcomputed tomography assessed bone metrics. Equivalent osteopenia occurs in Phe-restricted and untreated Pahenu2 control cohorts. In Phe unrestricted Pahenu2, creatine was without effect as bone density remained equivalent to Pahenu2 control cohorts. Alternatively, Phe-restricted Pahenu2 receiving creatine present increased bone density. We hypothesize small molecule dysregulation in untreated Pahenu2 disallows creatine utilization; therefore, osteopenia persisted. Dietary Phe restriction enables creatine utilization to enhance SSC osteoblast differentiation and improve in vivo bone density. PKU intervention singularly focused on Phe reduction enables residual disease including osteopenia and neurologic elements. Intervention concurrently addressing Phe homeostasis and energy dysregulation will improve disease elements refractory to standard of care Phe reduction mono-therapy

Chemotherapeutic Agents in Noncytotoxic Concentrations Increase Antigen Presentation by Dendritic Cells via an IL-12-Dependent Mechanism

Antineoplastic chemotherapeutic agents may indirectly activate dendritic cells (DCs) by inducing the release of danger signals from dying tumor cells. Whereas the direct cytotoxic or inhibitory effect of conventional chemotherapy on DCs has been reported, modulation of DC function by chemotherapeutic agents in low noncytotoxic concentrations has not yet been investigated. We have tested the effects of different classes of antineoplastic chemotherapeutic agents used in low noncytotoxic concentrations on the Ag-presenting function of DCs. We revealed that paclitaxel, doxorubicin, mitomycin C, and methotrexate up-regulated the ability of DCs to present Ags to Ag-specific T cells. Stimulation of DC function was associated with the up-regulation of expression of Ag-processing machinery components and costimulatory molecules on DCs, as well as increased IL-12p70 expression. However, the ability of DCs treated with paclitaxel, methotrexate, doxorubicin, and vinblastine to increase Ag presentation to Ag-specific T cells was abolished in DCs generated from IL-12 knockout mice, indicating that up-regulation of Ag presentation by DCs is IL-12-dependent and mediated by the autocrine or paracrine mechanisms. At the same time, IL-12 knockout and wild-type DCs demonstrated similar capacity to up-regulate OVA presentation after their pretreatment with low concentrations of mitomycin C and vincristine, suggesting that these agents do not utilize IL-12-mediated pathways in DCs for stimulating Ag presentation. These findings reveal a new mechanism of immunopotentiating activity of chemotherapeutic agents-a direct immunostimulatory effect on DCs (chemomodulation)-and thus provide a strong rationale for further assessment of low-dose chemotherapy given with DC vaccines for cancer treatment. The Journal of Immunology, 2009, 183: 137-144

Epithelial-like transport of mineral distinguishes bone formation from other connective tissues

We review unique properties of bone formation including current understanding of mechanisms of bone mineral transport. We focus on formation only; mechanism of bone degradation is a separate topic not considered. Bone matrix is compared to other connective tissues composed mainly of the same proteins, but without the specialized mechanism for continuous transport and deposition of mineral. Indeed other connective tissues add mechanisms to prevent mineral formation. We start with the epithelial-like surfaces that mediate transport of phosphate to be incorporated into hydroxyapatite in bone, or in its ancestral tissue, the tooth. These include several phosphate producing or phosphate transport-related proteins with special expression in large quantities in bone, particularly in the bone-surface osteoblasts. In all connective tissues including bone, the proteins that constitute the protein matrix are mainly type I collagen and γ−carboxylate-containing small proteins in similar molar quantities to collagen. Specialized proteins that regulate connective tissue structure and formation are surprisingly similar in mineralized and non-mineralized tissues. While serum calcium and phosphate are adequate to precipitate mineral, specialized mechanisms normally prevent mineral formation except in bone, where continuous transport and deposition of mineral occurs

Molecular Regulation of Bone Turnover in Juvenile Idiopathic Arthritis: Animal Models, Cellular Features and TNFα

We review the abnormal bone turnover that is the basis of idiopathic inflammatory or rheumatoid arthritis and bone loss, with emphasis on Tumor Necrosis Factor-alpha (TNFα)-related mechanisms. We review selected data on idiopathic arthritis in juvenile human disease, and discuss mouse models focusing on induction of bone resorbing cells by TNFα and Receptor Activator of Nuclear Factor kappa B Ligand (RANKL). In both humans and animal models, macrophage-derived cells in the joint, particularly in the synovium and periosteum, degrade bone and cartilage. Mouse models of rheumatoid arthritis share with human disease bone resorbing cells and strong relation to TNFα expression. In humans, differences in therapy and prognosis of arthritis vary with age, and results from early intervention for inflammatory cytokines in juvenile patients are particularly interesting. Mechanisms that contribute to inflammatory arthritis reflect, in large part, inflammatory cytokines that play minor roles in normal bone turnover. Changes in inflammatory cytokines, particularly TNFα, are many times larger, and presented in different locations, than cytokines that regulate normal bone turnover. Recent data from in vitro and mouse models include novel mechanisms described in differentiation of bone resorbing cells in inflammatory arthritis dependent on the Transient Receptor Potential Channel (TRPC) family of calcium channels. Low-molecular weight (MW) inhibitors of TRPC channels add to their potential importance. Associations with inflammatory arthritis unrelated to TNFα are briefly summarized as pointing to alternative mechanisms. We suggest that early detection and monoclonal antibodies targeting cytokines mediating disease progression deserves emphasis