Mechanism of action of amylin in bone.

Amylin is a 37 amino acid peptide produced mainly by beta-cells of the endocrine pancreas. Human amylin has 43% homology with human calcitonin gene-related peptide (CGRP) and 13% homology with human calcitonin (CT). Amylin and CGRP have been reported to have CT-like hypocalcemic activity in vivo. To investigate the role of amylin in bone, we examined the mechanisms of action of human amylin, CGRP, and CT in osteoclasts and osteoblasts. Both human amylin and CGRP inhibited 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3]- induced bone resorption in an organ culture system, and the potencies of the two peptides were similarly approximately 60-fold lower than that of human CT. Using a recently developed procedure for preparing large numbers of osteoclast-like multinucleated cells (MNCs) formed in co-cultures of mouse osteoblasts and bone marrow cells in the presence of 1 alpha,25(OH)2D3, we found that both human amylin and CGRP stimulated cAMP production in osteoclast-like MNCs, but only at 60-fold higher concentrations than human CT. Specific binding of [125I]-human CT to osteoclast-like MNCs was detected (dissociation constant, 3 x 10(-8) M; binding sites, 3 x 10(7) per cell). To displace the bound [125I]-human CT from osteoclast-like MNCs, about 170-fold higher concentrations of human amylin and CGRP were required. No specific bindings of [125I]-amylin and [125I]-CGRP to osteoclast-like MNCs could be detected. Human CGRP stimulated cAMP production both in established mouse osteoblast-like cells (KS-4) and in mouse primary osteoblast-like cells. Amylin was a weak agonist for cAMP production in KS-4 cells. The increment in cAMP production induced by CGRP and amylin was abolished by the addition of human CGRP(8-37), a selective antagonist for CGRP receptors. CT did not stimulate cAMP production in KS-4 cells. Amylin, but not CT, displaced the bound [125I]-human CGRP from rat brain membranes. These results indicate that amylin binds not only to CT receptors in osteoclast-like MNCs but also to CGRP receptors in osteoblasts. The relative potencies of these compounds to induce cAMP production was CT greater than amylin not equal to CGRP in osteoclast-like MNCs and CGRP greater amylin much greater than CT in osteoblast-like cells.     

A novel mutation of ALK2, L196P, found in the most benign case of fibrodysplasia ossificans progressiva activates BMP-specific intracellular signaling equivalent to a typical mutation, R206H

Naphthoquinone derivatives have been reported to possess various pharmacological activities, such as antiplatelet, anticancer, antifungal, and antiviral properties. In this study, we investigated the effects of a newly-synthesized naphthoquinone derivative, 2-decylamino-5,8-dimethoxy-1,4-naphthoquinone (2-decylamino-DMNQ), on VSMC proliferation and examined the molecular basis of the underlying mechanism. In a dose-dependent manner, 2-decylamino-DMNQ inhibited PDGF-stimulated VSMC proliferation with no apparent cytotoxic effect. While 2-decylamino-DMNQ did not affect PDGF-Rβ or Akt, it did inhibit the phosphorylation of Erk1/2 and PLCγ1 induced by PDGF. Moreover, 2-decylamino-DMNQ suppressed DNA synthesis through the arrest of cell cycle progression at the G₀/G₁ phase, including the suppression of pRb phosphorylation and a decrease in PCNA expression, which was related to the downregulation of cell cycle regulatory factors, such as cyclin D1/E and CDK 2/4. It was demonstrated that both U0126, an Erk1/2 inhibitor, and U73122, a PLCγ inhibitor, increased the proportion of cells in the G₀/G₁ phase of the cell cycle. Thus, these results suggest that 2-decylamino DMNQ has an inhibitory effect on PDGF-induced VSMC proliferation and the mechanism of this action is through cell cycle arrest at the G₀/G₁ phase. This may be a useful tool for studying interventions for vascular restenosis in coronary revascularization procedures and stent implantation.     

Constitutively activated ALK2 and increased SMAD1/5 cooperatively induce bone morphogenetic protein signaling in fibrodysplasia ossificans progressiva

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant disorder characterized by congenital malformation of the great toes and by progressive heterotopic bone formation in muscle tissue. Recently, a mutation involving a single amino acid substitution in a bone morphogenetic protein (BMP) type I receptor, ALK2, was identified in patients with FOP. We report here that the identical mutation, R206H, was observed in 19 Japanese patients with sporadic FOP. This mutant receptor, ALK2(R206H), activates BMP signaling without ligand binding. Moreover, expression of Smad1 and Smad5 was up-regulated in response to muscular injury. ALK2(R206H) with Smad1 or Smad5 induced osteoblastic differentiation that could be inhibited by Smad7 or dorsomorphin. Taken together, these findings suggest that the heterotopic bone formation in FOP may be induced by a constitutively activated BMP receptor signaling through Smad1 or Smad5. Gene transfer of Smad7 or inhibition of type I receptors with dorsomorphin may represent strategies for blocking the activity induced by ALK2(R206H) in FOP.     

A novel mutation of ALK2, L196P, found in the most benign case of fibrodysplasia ossificans progressiva activates BMP-specific intracellular signaling equivalent to a typical mutation, R206H

Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant congenital disorder characterized by progressive heterotopic ossification in muscle tissues. Constitutively activated mutants of a bone morphogenetic protein (BMP) receptor, ALK2, have been identified in patients with FOP. Recently, a novel ALK2 mutation, L196P, was found in the most benign case of FOP reported thus far. In the present study, we examined the biological activities of ALK2(L196P) in vitro. Over-expression of ALK2(L196P) induced BMP-specific activities, including the suppression of myogenesis, the induction of alkaline phosphatase activity, increased BMP-specific luciferase reporter activity, and increased phosphorylation of Smad1/5 but not Erk1/2 or p38. The activities of ALK2(L196P) were higher than those of ALK2(G356D), another mutant ALK2 allele found in patients with FOP and were equivalent to those of ALK2(R206H), a typical mutation found in patients with FOP. ALK2(L196P) was equally or more resistant to inhibitors in comparison to ALK2(R206H). These findings suggest that ALK2(L196P) is an activated BMP receptor equivalent to ALK2(R206H) and that ALK2(L196P) activity may be suppressed in vivo by a novel molecular mechanism in patients with this mutation.     

Health Outcomes of Gastric Bypass Patients Compared to Nonsurgical,Nonintervened Severely Obese

Favorable health outcomes at 2 years postbariatric surgery have been reported. With exception of the Swedish Obesity Subjects (SOS) study, these studies have been surgical case series, comparison of surgery types, or surgery patients compared to subjects enrolled in planned nonsurgical intervention. This study measured gastric bypass effectiveness when compared to two separate severely obese groups not participating in designed weight‐loss intervention. Three groups of severely obese subjects (N = 1,156, BMI ≥ 35 kg/m²) were studied: gastric bypass subjects (n = 420), subjects seeking gastric bypass but did not have surgery (n = 415), and population‐based subjects not seeking surgery (n = 321). Participants were studied at baseline and 2 years. Quantitative outcome measures as well as prevalence, incidence, and resolution rates of categorical health outcome variables were determined. All quantitative variables (BMI, blood pressure, lipids, diabetes‐related variables, resting metabolic rate (RMR), sleep apnea, and health‐related quality of life) improved significantly in the gastric bypass group compared with each comparative group (all P < 0.0001, except for diastolic blood pressure and the short form (SF‐36) health survey mental component score at P < 0.01). Diabetes, dyslipidemia, and hypertension resolved much more frequently in the gastric bypass group than in the comparative groups (all P < 0.001). In the surgical group, beneficial changes of almost all quantitative variables correlated significantly with the decrease in BMI. We conclude that Roux‐en‐Y gastric bypass surgery when compared to severely obese groups not enrolled in planned weight‐loss intervention was highly effective for weight loss, improved health‐related quality of life, and resolution of major obesity‐associated complications measured at 2 years.     

Mutations in CD96, a member of the immunoglobulin superfamily, cause a form of the C (Opitz trigonocephaly) syndrome

The C syndrome is characterized by trigonocephaly and associated anomalies, such as unusual facies, psychomotor retardation, redundant skin, joint and limb abnormalities, and visceral anomalies. In an individual with the C syndrome who harbors a balanced chromosomal translocation, t(3;18)(q13.13;q12.1), we discovered that the TACTILE gene for CD96, a member of the immunoglobulin superfamily, was disrupted at the 3q13.3 breakpoint. In mutation analysis of nine karyotypically normal patients given diagnoses of the C or C-like syndrome, we identified a missense mutation (839C-->T, T280M) in exon 6 of the CD96 gene in one patient with the C-like syndrome. The missense mutation was not found among 420 unaffected Japanese individuals. Cells with mutated CD96 protein (T280M) lost adhesion and growth activities in vitro. These findings indicate that CD96 mutations may cause a form of the C syndrome by interfering with cell adhesion and growth.     

Characterization of dynamic three-dimensional strain fields in the canine radius

This note describes a method to approximate the 3-D mechanical environment of a long bone during a normal daily activity. Our specific goal was to characterize the temporal and spatial strain distributions in the mid-shaft region of the canine radius during gait. Direct measurement of strains along the entire surface of in vivo bone is not feasible, so we employed a combination of experimental measurements and numerical interpolation techniques to approximate the time-varying longitudinal strain distribution. Using standard in vivo strain gauging techniques, we measured dynamic strains at nine locations (three locations on each of three cross sections, data pooled from two experimental animals) on the canine radius during trotting gait. These in vivo strain measurements were then used to approximate the time course of the strain field for the entire radius mid-shaft region using a 3-D numerical interpolation scheme using finite element basis functions. Despite limitations in the present implementation of the method, the results show that there are considerable time-dependent variations in the strain distribution occurring at different transverse sections along the length of the diaphysis with substantial anteroposterior bending and rotation of neutral axis locations during the gait cycle.     

Midkine as a novel target for antibody therapy in osteosarcoma

Osteosarcoma is a malignant tumor with poor prognosis, and lack of accurate prognostic factors is one of the reasons that make this tumor difficult to cure. The heparin-binding growth factor, midkine is involved in generation and progression of many types of tumors. However, the relationship between midkine and osteosarcoma has been unclear. We show here that midkine is overexpressed in osteosarcoma and the level of midkine expression is correlated with prognosis (P<0.05; log-rank test). Treatment with functional antibodies against midkine suppresses growth of osteosarcoma cell lines, 9N2, 3N1, Saos-2, and NOS-1, to 25-65% of untreated controls. Our results suggest that midkine is useful as a prognostic marker, and is a candidate therapeutic target for osetosarcomas.