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While menin plays an important role in preventing T-cell dysfunction, such as senescence and exhaustion, the regulatory mechanisms remain unclear. We found that menin prevents the induction of dysfunction in activated CD8 T cells by restricting the cellular metabolism. mTOR complex 1 (mTORC1) signaling, glycolysis, and glutaminolysis are augmented by menin deficiency. Rapamycin treatment prevents CD8 T-cell dysfunction in menin-deficient CD8 T cells. Limited glutamine availability also prevents CD8 T-cell dysfunction induced by menin deficiency, and its inhibitory effect is antagonized by α-ketoglutarate (α-KG), an intermediate metabolite of glutaminolysis. α-KG-dependent histone H3K27 demethylation seems to be involved in the dysfunction in menin-deficient CD8 T cells. We also found that α-KG activates mTORC1-dependent central carbon metabolism. These findings suggest that menin maintains the T-cell functions by limiting mTORC 1 activity and subsequent cellular metabolism. [ABSTRACT FROM AUTHOR]

C-C chemokine receptor 5 (CCR5) is a co-receptor of HIV. Epidemiological findings suggest that the functional loss of CCR5 is correlated with a lower incidence of bone-destructive diseases as well as of HIV transmission. However, it is not clear whether CCR5 is involved in regulation of the function of bone cells, in addition to that of immune cells. Here we show that blockade of CCR5 using specific antibodies impairs human osteoclast function in vitro. Ccr5- deficient (Ccr5-/-) mice presented with dysfunctional osteoclasts and were resistant to osteoporosis induced by receptor activator of nuclear factor kappa-B ligand (RANKL), which triggers osteoporosis independently of inflammatory and immunomodulatory pathways. Furthermore, Ccr5 deficiency impairs the cellular locomotion and bone-resorption activity of osteoclasts, which is associated with the disarrangement of podosomes and adhesion complex molecules including Pyk2. Overall, the data provides evidence that CCR5 has an essential role in bone-destructive conditions through the functional regulation of osteoclasts. [ABSTRACT FROM AUTHOR]

Abstract  Recombinant human bone morphogenetic protein (rhBMP) is a promising therapeutic cytokine for the induction of bone formation, but a weak response in humans remains a major hurdle in its therapeutic application. We have previously reported an rhBMP-2-induced increase in the bone mass of mice receiving systemic rolipram, a specific inhibitor of phosphodiesterase-4. To overcome the side effects of systemic administration of rolipram, we examined the effects of its local release. Polyethylene glycol discs were used as a delivery system. The discs were impregnated with rhBMP-2 and rolipram and implanted into the dorsal muscle pouches in mice. Bone formation was assessed by measuring the bone mineral content (BMC) of the formed bone. First, to determine the optimal dose of rolipram, we added 0–5000 nmol rolipram and 5 μg rhBMP-2 to the pellets and found that 500 nmol rolipram was the most effective concentration for inducing bone formation after 4 weeks. Second, to examine the time course of bone formation, we implanted 5 μg rhBMP-2 with 0 or 500 nmol rolipram and killed mice 5, 7, 10, 14, or 21 days after implantation. Bone formation was accelerated in the rolipram group. Finally, to determine the rolipram-induced increase in the effect of BMP, BMC obtained after treatment with 5 μg rhBMP-2 and 500 nmol rolipram was compared with that obtained after treatment with 5–9 μg rhBMP-2 without rolipram, 4 weeks after implantation. The results indicated that 500 nmol rolipram enhanced the effect of rhBMP-2 by almost 1.5-fold. In summary, locally released rolipram enhanced the capacity of rhBMP-2 to induce bone formation, an effect previously reported with systemic administration. These findings may decrease the cost and increase the efficacy of rhBMP-2 treatment. [ABSTRACT FROM AUTHOR]

Abstract  One problem associated with clinical application of CHO-derived recombinant human bone morphogenetic protein (C-BMP-2) is its high cost due to the need for use of high doses. To solve this problem, Escherichia coli-derived BMP-2 (E-BMP-2) has been examined using the technique of molecular unfolding and refolding. However, it is unclear whether the characteristics of E-BMP-2 are appropriate for clinical application. In this study, we examined the biological activity of E-BMP-2 and its heat tolerance in in vitro and in vivo systems. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) confirmed the high purity of E-BMP-2. E-BMP-2-induced alkaline phosphatase expression in osteoprogenitor cells (C2C12, ST2, and primary murine calvarial osteoblast cells) was dose-dependent, and consistently elicited ectopic new ossicles of significant size in mice, also in dose-dependent fashion. In addition, E-BMP-2 induced phosphorylation of Smad1/5/8 and mRNA expression of osteoblastic differentiation markers to the same extent as C-BMP-2. On the other hand, when E-BMP-2 was exposed to increasing heat over time, its bone-inducing capacity was maintained until reaching 70°C for 2 h or 90°C for 15 min. Thus, E-BMP-2 will exhibit a decrease in activity with the sterilization procedures required prior to use in surgery. These findings indicate that the biological capacity and heat stability of E-BMP-2 are almost equivalent to those of currently available C-BMP-2, and suggest that E-BMP-2 might, thus, solve current problems of cost impeding routine clinical use of rhBMP-2. [ABSTRACT FROM AUTHOR]

Abstract  We present experimental results indicating involvement of cyclic AMP (cAMP)-mediated signaling in bone morphogenetic protein (BMP)-induced osteoblastic gene expression at the transcriptional level by luciferase activity assay in C2C12 cells using the promoter sequence of the Id1 gene, an early-response gene to BMPs, which contains both a BMP-responsive element (BRE) and a cAMP-response element (CRE). In cells transfected with luciferase gene driven by wild-type Id1 promoter, treatment with BMP-4 increased luciferase expression, which was further enhanced by the addition of dibutyryl cAMP (dbcAMP). This dbcAMP-enhanced luciferase expression was significantly suppressed when the CRE site in the Id1 promoter was replaced by mutated CRE or endogenous CRE-binding protein (CREB) was knocked down by transfection of CREB RNAi. Pretreatment of cells with protein kinase A (PKA) inhibitor, H89, also dramatically reduced dbcAMP-enhanced luciferase expression. Immunoprecipitation assay showed phosphorylated-Smad1/5/8, phosphorylated-CREB, and CREB-binding protein (CBP) formed the transcriptional complex. These data indicate that cAMP-PKA/CREB/CRE signaling potentially enhances BMP-induced transcription through the BRE in the promoter of the BMP-responsive gene through a PKA-mediated pathway. [ABSTRACT FROM AUTHOR]

Abstract  The promotion of osteoblastic differentiation by bone morphogenetic proteins (BMPs) is accelerated by chemical compounds that increase the intracellular concentration of cyclic 3′,5′-adenosine monophosphate (cAMP). cAMP is synthesized from adenosine triphosphate (ATP) by adenyl cyclase and degraded by phosphodiesterase (PDE) family enzymes. Inhibition of PDEs leads to prolonged accumulation of cAMP within cells and Camp-mediated reactions. Rolipram, a specific inhibitor of PDE4, is a compound effective in inducing osteoblastic differentiation. Four PDE4 family members are transcribed from four distinct genes (4A, 4B, 4C, and 4D). Expression of PDE4A and PDE4D has been observed in osteoblastic cells. We identified PDE4D splicing variants that expressed in ST2 or primary calvarial osteoblasts by rapid amplification of the 5′-ends of cDNA when they were cultured with BMP. PDE4D9 mRNA was identified from ST2, and PDE4D1 and −4D2 mRNAs were identified from primary calvarial osteoblasts. Expression of these three variants of PDE4D mRNA was found in ST2, MC3T3-E1, C3H10T1/2, C2C12, and primary calvarial osteoblasts by RT-PCR, but not PDE4D1 or −4D2 in ST2 or PDE4D2 in MC3T3-E1. Expression of these three variants was detectable in brain, heart, lung, liver, kidney, placenta, and femur, and was thus ubiquitous. Purified recombinant PDE4D9 protein exhibited phosphodiesterase activity, which degraded cAMP to AMP, and this activity was inhibited by rolipram. These findings suggest that PDE4D1, −2, and −9 play some roles in bone formation. [ABSTRACT FROM AUTHOR]

This study was designed to investigate effects of heat on the bone-inducing activity of recombinant human bone morphogenetic protein (rhBMP)-2. rhBMP-2 samples were heated at 50, 70, 90, or 100°C for 15min, or 1, 2, 4, or 8h, or autoclaved at 120°C for 15min. The bone-inducing activity of the rhBMP-2 before and after heating was assayed in in vivo and in vitro systems. For the in vivo assay, 5µg rhBMP-2 samples were impregnated into porous collagen disks (6mm in diameter, 1mm thickness), freeze dried, and implanted into the back muscles of ddY mice. Three weeks later, the implant was harvested from the host and examined for ectopic new bone tissue by radiography. The new bone mass was quantified by single-energy X-ray absorptiometry. The in vitro activity of the rhBMP-2 was assayed by adding the BMP sample at a concentration of 100ng/ml to cultures of MC3T3-E1 cells. After 48h, the alkaline phosphatase activity was measured. After heating at 50° or 70°C, no significant reduction in bone-inducing activity was noted in either in vivo or in vitro assay systems unless the protein was exposed to sustained heat at 70°C for 8h, based on in vitro assay data. However, heating above 90°C and for longer periods led to a decrease in the biological activity of the rhBMP-2 in a time- and temperature-dependent manner. rhBMP-2 was rendered inactive when exposed to temperatures at or in excess of 120°C. [ABSTRACT FROM AUTHOR]