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A major cause of osteoporosis is impaired coupled bone formation. Mechanistically, both osteoclast-derived and bone-derived growth factors have been previously implicated. Here, we hypothesize that the release of bone calcium during osteoclastic bone resorption is essential for coupled bone formation. Osteoclastic resorption increases interstitial fluid calcium locally from the normal 1.8 mM up to 5 mM. MC3T3-E1 osteoprogenitor cells, cultured in a 3.6 mM calcium medium, demonstrated that calcium signaling stimulated osteogenic cell proliferation, differentiation, and migration. Calcium channel knockdown studies implicated calcium channels, Cav1.2, store-operated calcium entry (SOCE), and calcium-sensing receptor (CaSR) in regulating bone cell anabolic activities. MC3T3–E1 cells cultured in a 3.6 mM calcium medium expressed increased gene expression of Wnt signaling and growth factors platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and bone morphogenic protein-2 (BMP 2). Our coupling model of bone formation, the receptor activator of nuclear factor-κΒ ligand (RANKL)-treated mouse calvaria, confirmed the role of calcium signaling in coupled bone formation by exhibiting increased gene expression for osterix and osteocalcin. Critically, dual immunocytochemistry showed that RANKL treatment increased osterix-positive cells and increased fluorescence intensity of Cav1.2 and CaSR protein expression per osterix-positive cell. The above data established that calcium released by osteoclasts contributed to the regulation of coupled bone formation. CRISPR/Cas-9 knockout of Cav1.2 in osteoprogenitor cells cultured in basal calcium medium caused a >80% decrease in the expression of downstream osteogenic genes, emphasizing the large magnitude of the effect of calcium signaling. Thus, calcium signaling is a major regulator of coupled bone formation.

Acute liver injury is a common disease without effective therapy in humans. We sought to evaluate a combination therapy of insulin-like growth factor 1 (IGF-I) and BTP-2 in a mouse liver injury model induced by lipopolysaccharide (LPS). We chose this model because LPS is known to increase the expression of the transcription factors related to systemic inflammation (i.e., NFκB, CREB, AP1, IRF 3, and NFAT), which depends on calcium signaling. Notably, these transcription factors all have pleiotropic effects and account for the other observed changes in tissue damage parameters. Additionally, LPS is also known to increase the genes associated with a tissue injury (e.g., NGAL, SOD, caspase 3, and type 1 collagen) and systemic expression of pro-inflammatory cytokines. Finally, LPS compromises vascular integrity. Accordingly, IGF-I was selected because its serum levels were shown to decrease during systemic inflammation. BTP-2 was chosen because it was known to decrease cytosolic calcium, which is increased by LPS. This current study showed that IGF-I, BTP-2, or a combination therapy significantly altered and normalized all of the aforementioned LPS-induced gene changes. Additionally, our therapies reduced the vascular leakage caused by LPS, as evidenced by the Evans blue dye technique. Furthermore, histopathologic studies showed that IGF-I decreased the proportion of hepatocytes with ballooning degeneration. Finally, IGF-I also increased the expression of the hepatic growth factor (HGF) and the receptor for the epidermal growth factor (EGFR), markers of liver regeneration. Collectively, our data suggest that a combination of IGF-I and BTP-2 is a promising therapy for acute liver injury.

Acute lung injury (ALI) afflicts approximately 200,000 patients annually and has a 40% mortality rate. The COVID-19 pandemic has massively increased the rate of ALI incidence. The pathogenesis of ALI involves tissue damage from invading microbes and, in severe cases, the overexpression of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). This study aimed to develop a therapy to normalize the excess production of inflammatory cytokines and promote tissue repair in the lipopolysaccharide (LPS)-induced ALI. Based on our previous studies, we tested the insulin-like growth factor I (IGF-I) and BTP-2 therapies. IGF-I was selected, because we and others have shown that elevated inflammatory cytokines suppress the expression of growth hormone receptors in the liver, leading to a decrease in the circulating IGF-I. IGF-I is a growth factor that increases vascular protection, enhances tissue repair, and decreases pro-inflammatory cytokines. It is also required to produce anti-inflammatory 1,25-dihydroxyvitamin D. BTP-2, an inhibitor of cytosolic calcium, was used to suppress the LPS-induced increase in cytosolic calcium, which otherwise leads to an increase in proinflammatory cytokines. We showed that LPS increased the expression of the primary inflammatory mediators such as toll like receptor-4 (TLR-4), IL-1β, interleukin-17 (IL-17), TNF-α, and interferon-γ (IFN-γ), which were normalized by the IGF-I + BTP-2 dual therapy in the lungs, along with improved vascular gene expression markers. The histologic lung injury score was markedly elevated by LPS and reduced to normal by the combination therapy. In conclusion, the LPS-induced increases in inflammatory cytokines, vascular injuries, and lung injuries were all improved by IGF-I + BTP-2 combination therapy.

This study was undertaken to test two therapies for acute kidney injury (AKI) prevention, IGF-1, which is renal protective, and BTP-2, which is a calcium entry (SOCE) inhibitor. We utilized lipopolysaccharide (LPS) IP, as a systemic model of AKI and studied in five groups of animals. Three experiments showed that at 7 days: (1) LPS significantly reduced serum IGF-1 and intramuscular IGF-I in vivo gene therapy rescued this deficiency. (2) Next, at the 7-day time point, our combination therapy, compared to the untreated group, caused a significant increase in survival, which was noteworthy because all of the untreated animals died in 72 h. (3) The four pathways associated with inflammation, including (A) increase in cytosolic calcium, (B) elaboration of proinflammatory cytokines, (C) impairment of vascular integrity, and (D) cell injury, were adversely affected in renal tissue by LPS, using a sublethal dose of LPS. The expression of several genes was measured in each of the above pathways. The combined therapy of IGF-1 and BTP-2 caused a favorable gene expression response in all four pathways. Our current study was an AKI study, but these pathways are also involved in other types of severe inflammation, including sepsis, acute respiratory distress syndrome, and probably severe coronavirus infection.