BackgroundBone morphogenic proteins (BMPs) play a key role in bone formation. Consequently, it was expected that topical application of recombinant human (rh)BMP-2 and rhBMP-7 would improve the healing of complex fractures. However, up to 36% of fracture patients do not respond to this therapy. There are hints that a systemic increase in transforming growth factor β1 (TGFβ1) interferes with beneficial BMP effects. Therefore, in the present work we investigated the influence of rhTGFβ1 on rhBMP signaling in primary human osteoblasts, with the aim of more specifically delineating the underlying regulatory mechanisms.MethodsBMP signaling was detected by adenoviral Smad-binding-element-reporter assays. Gene expression was determined by reverse transcription polymerase chain reaction (RT-PCR) and confirmed at the protein level by western blot. Histone deacetylase (HDAC) activity was determined using a test kit. Data sets were compared by one-way analysis of variance.ResultsOur findings showed that Smad1/5/8-mediated rhBMP-2 and rhBMP-7 signaling is completely blocked by rhTGFβ1. We then investigated expression levels of genes involved in BMP signaling and regulation (for example, Smad1/5/8, TGFβ receptors type I and II, noggin, sclerostin, BMP and activin receptor membrane bound inhibitor (BAMBI), v-ski sarcoma viral oncogene homolog (Ski), Ski-related novel protein N (SnoN) and Smad ubiquitination regulatory factors (Smurfs)) and confirmed the expression of regulated genes at the protein level. Smad7 and SnoN were significantly induced by rhTGFβ1 treatment while expression of Smad1, Smad6, TGFβRII and activin receptor-like kinase 1 (Alk1) was reduced. Elevated SnoN expression was accompanied by increased HDAC activity. Addition of an HDAC inhibitor, namely valproic acid, fully abolished the inhibitory effect of rhTGFβ1 on rhBMP-2 and rhBMP-7 signaling.ConclusionsrhTGFβ1 effectively blocks rhBMP signaling in osteoblasts. As possible mechanism, we postulate an induction of SnoN that increases HDAC activity and thereby reduces the expression of factors required for efficient BMP signaling. Thus, inhibition of HDAC activity may support bone healing during rhBMP therapy in patients with elevated TGFβ serum levels.
Promotion of rhBMP2 and rhBMP7 for the routine use to support fracture healing has been hampered by high costs, safety concerns and reasonable failure rates, imposing restrictions in its clinical use. Since there is little debate regarding its treatment potential, there is rising need for a better understanding of the mode of action of these BMPs to overcome its drawbacks and promote more efficacious treatment strategies for bone regeneration. Recently, BMP9, owing to its improved osteogenic potential, is gaining attention as a promising therapeutic alternative. Our study aimed at identifying specific gene expression patterns which may predict and explain individual responses to rhBMP7 and rhBMP9 treatments. Therefore, we investigated the effect of rhBMP7 and rhBMP9 on primary human osteoblasts from 110 donors and corresponding THP-1-derived osteoclasts. This was further compared with each other and our reported data on rhBMP2 response. Based on the individual donor response, we found three donor groups profiting from rhBMP treatment either directly via stimulation of osteoblast function or viability and/or indirectly via inhibition of osteoclasts. The response on rhBMP7 treatment correlated with expression levels of the genes BAMBI, SOST, Noggin, Smad4 and RANKL, while the response of rhBMP9 correlated to the expression levels of Alk6, Endoglin, Smurf1, Smurf2, SOST and RANKL in these donors. Noteworthy, rhBMP9 treatment showed significantly increased osteogenic activity (AP activity and Smad nuclear translocation) when compared to the two clinically used rhBMPs. Based on patient's respective expression profiles, clinical application of rhBMP9 either solely or in combination with rhBMP2 and/or rhBMP7 can become a promising new approach to fit the patient's needs to promote fracture healing.
OBJECTIVEThe study investigated the effect of angiotensin receptor blockers (ARB) on glucose homeostasis and inflammatory parameters in patients with impaired glucose tolerance (IGT).RESEARCH DESIGN AND METHODSWe prospectively studied the insulin sensitivity index (ISI) and homeostasis model assessment–insulin resistance (HOMA-IR) in 13 obese males with IGT and in 13 matched control subjects with normal glucose tolerance (NGT) during hyperglycemic testing over 90 min. Adiponectin, retinol-binding protein 4 (RBP4), and high-sensitive C-reactive protein (hsCRP) were analyzed. Measurements were performed at baseline and after a 4-week treatment with 160 mg/day valsartan. The results of the IGT and NGT groups were compared.RESULTSAt baseline, HOMA-IR (IGT 4.1 ± 3 vs. NGT 2.3 ± 1.0, P < 0.01), hsCRP (IGT 3.9 ± 1.9 vs. NGT 1.8 ± 1 mg/l, P < 0.05), and RBP4 (IGT 27.1 ± 2.1 vs. NGT 24.0 ± 2.0 ng/ml, P < 0.05) were significantly higher, whereas ISI (IGT 1.5 ± 0.9 vs. NGT 1.8 ± 1.2, P < 0.05) and plasma adiponectin (IGT 3.2 ± 0.9, NGT 5.2 ± 2.4 μg/ml, P < 0.05) were significantly lower in the IGT group compared with the NGT group. Under ARB, there was an increase in both groups of adiponectin (IGT 4.1 ± 1.9 μg/ml, NGT 6.3 ± 2.9 μg/ml, P < 0.05) and an increase in ISI (IGT 1.5 ± 0.9 to 2.3 ± 1 μg/ml, NGT 1.8 ± 1 to 2.5 ± 2 μg/ml, P < 0.05). HOMA-IR (4.1 ± 3 to 2.6 ± 2; P < 0.01), hsCRP (3.9 ± 1.9 to 1.8 ± 1 mg/l, P < 0.05), and RBP4 (27.1 ± 2.1 to 22.1 ± 1.8 ng/ml, P < 0.01) decreased significantly in the IGT group.CONCLUSIONSInsulin sensitivity and associated inflammatory factors improve under ARB in IGT patients.
Affecting more than 230,000,000 patients, diabetes mellitus is one of the most frequent metabolic disorders in developed countries. Among other complications, diabetic patients have an increased fracture risk and show delayed fracture healing. During the disease progression, these patients' blood glucose and insulin levels vary significantly. Thus, the aim of this study was to analyze the effects of glucose and insulin on primary human osteoblasts. Although, in the presence of insulin and glucose, proliferation of osteoblasts was increased (1.2- to 1.7-fold), their alkaline phosphatase activity and, consequently, production of mineralized matrix were significantly reduced down to 55 % as compared to control cells (p < 0.001). Interestingly, the observed effects were mainly due to stimulation with insulin. Increase in glucose did not alter osteoblasts' function significantly but further enhanced the effects of insulin. Expression of active and total transforming growth factor beta (TGF-β) was increased by glucose and insulin. Stimulation with both glucose and insulin induced gene expression changes (e.g., osteocalcin, Runx2, Satb2, or Stat1) comparable to treatment with recombinant TGF-β(1), further indicating osteoblasts' dysfunction. Inhibition of TGF-β signaling completely abolished the negative effects of glucose and insulin. In summary, glucose and insulin treatment causes osteoblast dysfunction, which is accompanied by an increased TGF-β expression. Blocking TGF-β signaling abrogates the functional loss observed in glucose- and insulin-treated osteoblasts, thus identifying TGF-β as a key regulator. Therefore, increased TGF-β expression during diabetes may be a feasible pathogenic mechanism underlying poor bone formation in uncontrolled diabetes mellitus.
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