Harry C. Blair

This is the first study to assess directly the effects of very low concentrations of ACTH _1–24 , picomolar to micromolar, on regulation of bone growth. It shows effects on bone mass in the femoral heads and by labeling bone formation in rabbits in vitro, and effect on key proteins related to bone growth mechanisms in cell preparations of human bone-forming cells in vitro.

Bone formation and resorption are mediated by an epithelial‐like cell layer on bone. Formation or resorption requires active transport that depends on aerobic glycolysis, ATP, and acid transport. Metabolic activity of bone cells during matrix formation or removal is so high that the cells autolyze rapidly after cell death. Mineralization of bone matrix uses import of phosphate by sodium‐phosphate cotransport, supported by the Na ⁺ /K ⁺ ATPase. Glucose is the main energy source; ATP is exported to generate phosphate for hydroxyapatite in the bone matrix. Mechanism of export is not established, but phosphate is generated at least in part via phosphatase/pyrophosphatase activity including the tissue nonspecific alkaline phosphatase (TNAP) and ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2). Ca ²⁺ is imported by paracellular transport. Protons, generated in producing hydroxyapatite, are exported by apical H ⁺ /Cl ⁻ exchangers ClC3 and ClC5, and basolateral Na ⁺ /H ⁺ exchange. In bone resorption, ATP‐dependent acid transport, the reverse of acid transport in bone formation, is essential. This uses the vacuolar‐type H ⁺ ATPase linked to Cl ⁻ transport via a ClC family H ⁺ /Cl ⁻ exchanger, ClC7, and a Cl ⁻ channel. Other transporters contributing include carbonic anhydrase and chloride‐bicarbonate exchange to replace H ⁺ equivalents exported for bone resorption. New and Noteworthy: This focused short review considers the relationship of oxidative phosphorylation to acid transport in bone formation and resorption, processes with very high metabolic activity for storage or removal of phosphate, calcium and acid equivalents.

Arthritis leads to bone erosion due to an imbalance between osteoclast and osteoblast function. Our prior investigations revealed that the Ca ²⁺ -selective ion channel, Orai1, is critical for osteoclast maturation. Here, we show that the small-molecule ELP-004 preferentially inhibits transient receptor potential canonical (TRPC) channels. While ELP-004 minimally affected physiological RANKL-induced osteoclast maturation in murine bone marrow– and spleen-derived myeloid cells (BMSMCs) and human PBMC-derived cells, it potently interfered with osteoclast maturation driven by TNFα or LTB4. The contribution of TRPC channels to osteoclastogenesis was examined using BMSMCs derived from TRPC4 ^−/− or TRPC(1–7) ^−/− mice, again revealing preferential interference with osteoclastogenesis driven by proinflammatory cytokines. ELP-004 also reduced bone erosion in a mouse model of rheumatoid arthritis. These investigations reveal TRPC channels as critical mediators of inflammatory bone erosion and provide insight into the major target of ELP-004, a drug currently in preclinical testing as a therapeutic for inflammatory arthritis.

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.

This study provides a detailed understanding of the preclinical pharmacokinetics and metabolism of ELP‐004, an osteoclast inhibitor in development for the treatment of bone erosion. Current treatments for arthritis, including biological disease‐modifying antirheumatic drugs, are not well‐tolerated in a substantial subset of arthritis patients and are expensive; therefore, new treatments are needed. Pharmacokinetic parameters of ELP‐004 were tested with intravenous, oral, and subcutaneous administration and found to be rapidly absorbed and distributed. We found that ELP‐004 was non‐mutagenic, did not induce chromosome aberrations, non‐cardiotoxic, and had minimal off‐target effects. Using in vitro hepatic systems, we found that ELP‐004 is primarily metabolized by CYP1A2 and CYP2B6 and predicted metabolic pathways were identified. Finally, we show that ELP‐004 inhibits osteoclast differentiation without suppressing overall T‐cell function. These preclinical data will inform future development of an oral compound as well as in vivo efficacy studies in mice.

We report the discovery that the ecto-nucleotide pyrophosphatase/phosphodiesterase, ENPP2, is an important regulator of early differentiation of bone-forming osteoblasts.

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.

This work for the first time showed that specific enzymes in bone mineral transport, and matrix synthesis proteins, in the epithelial-like bone-forming cell layer are downregulated with aging. Results were compared using cells extracted from long bones of young and old mice, or in essentially uniform osteoblasts differentiated from stromal stem cells in vitro. The age effect showed memory in the stromal stem cells, a remarkable finding.

The calcium-selective ion channel Orai1 has a complex role in bone homeostasis, with defects in both bone production and resorption detected in Orai1 germline knock-out mice. To determine whether Orai1 has a direct, cell-intrinsic role in osteoblast differentiation and function, we bred Orai1 flox/flox (Orai1^fl/fl) mice with Runx2-cre mice to eliminate its expression in osteoprogenitor cells. Interestingly, Orai1 was expressed in a mosaic pattern in Orai1^fl/fl-Runx2-cre bone. Specifically, antibody labeling for Orai1 in vertebral sections was uniform in wild type animals, but patchy regions in Orai1^fl/fl-Runx2-cre bone revealed Orai1 loss while in other areas expression persisted. Nevertheless, by micro-CT, bones from Orai1^fl/fl-Runx2-cre mice showed reduced bone mass overall, with impaired bone formation identified by dynamic histomorphometry. Cortical surfaces of Orai1^fl/fl-Runx2-cre vertebrae however exhibited patchy defects. In cell culture, Orai1-negative osteoblasts showed profound reductions in store-operated Ca²⁺ entry, exhibited greatly decreased alkaline phosphatase activity, and had markedly impaired substrate mineralization. We conclude that defective bone formation observed in the absence of Orai1 reflects an intrinsic role for Orai1 in differentiating osteoblasts.