Recombinant Human Bone Morphogenetic Protein 9 (rhBMP9) Induced Osteoblastic Behaviour on a Collagen Membrane Compared With rhBMP2

BACKGROUND Bone morphogenetic protein 9 (BMP9) has previously been characterized as one of the most osteogenic growth factors of the BMP-family, however, up until now, these experiments have only been demonstrated using adenovirus-transfection experiments (gene therapy). With the recent development of recombinant human (rh)BMP9, the aim of the present study was to investigate its osteopromotive potential versus rhBMP2 when loaded onto a collagen membrane. METHODS ST2 stromal bone marrow cells were seeded onto 1)control; 2)rhBMP2-low(10ng/ml); 3)rhBMP2-high(100ng/ml); 4)rhBMP9-low(10ng/ml); and 5)rhBMP9-high(100ng/ml) porcine collagen membranes. Groups were then compared for cell adhesion at 8 hours, cell proliferation at 1, 3 and 5 days real-time PCR at 3 and 14 days for genes encoding Runx2, alkaline phosphatase(ALP) and bone sialoprotein(BSP) at 3 and 14 days and alizarin red staining at 14 days. RESULTS While rhBMP2 and rhBMP9 demonstrated little effects on cell attachment and proliferation, pronounced increases were observed on osteoblast differentiation. It was found that all groups significantly induced ALP mRNA levels at 3 days and BSP levels at 14 days, however rhBMP9-high demonstrated significantly higher values when compared to all other groups for ALP levels (5-fold increase at 3 days and 2-fold increase at 14 days). Alizarin red staining further revealed that both concentrations of rhBMP9 induced up to 3-fold more staining when compared to rhBMP2. CONCLUSION These results indicate that the combination of collagen membranes with rhBMP9 significantly induced significantly higher ALP mRNA expression and alizarin red staining when compared to rhBMP2. These findings suggest that rhBMP9 may be a suitable growth factor for future regenerative procedures in bone biology.

Retinoic acid receptor beta and angiopoietin-like protein 1 are involved in the regulation of human androgen biosynthesis

Androgens are essential for sexual development and reproduction. However, androgen regulation in health and disease is poorly understood. We showed that human adrenocortical H295R cells grown under starvation conditions acquire a hyperandrogenic steroid profile with changes in steroid metabolizing enzymes HSD3B2 and CYP17A1 essential for androgen production. Here we studied the regulatory mechanisms underlying androgen production in starved H295R cells. Microarray expression profiling of normal versus starved H295R cells revealed fourteen differentially expressed genes; HSD3B2, HSD3B1, CYP21A2, RARB, ASS1, CFI, ASCL1 and ENC1 play a role in steroid and energy metabolism and ANGPTL1, PLK2, DUSP6, DUSP10 and FREM2 are involved in signal transduction. We discovered two new gene networks around RARB and ANGPTL1, and show how they regulate androgen biosynthesis. Transcription factor RARB stimulated the promoters of genes involved in androgen production (StAR, CYP17A1 and HSD3B2) and enhanced androstenedione production. For HSD3B2 regulation RARB worked in cooperation with Nur77. Secretory protein ANGPTL1 modulated CYP17A1 and DUSP6 expression by inducing ERK1/2 phosphorylation. By contrast, our studies revealed no evidence for hormones or cell cycle involvement in regulating androgen biosynthesis. In summary, these studies establish a firm role for RARB and ANGPTL1 in the regulation of androgen production in H295R cells.

Mutation Update of the CLCN5 Gene Responsible for Dent Disease 1

Dent disease is a rare X-linked tubulopathy characterized by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis and/or nephrolithiasis, progressive renal failure, and variable manifestations of other proximal tubule dysfunctions. It often progresses over a few decades to chronic renal insufficiency, and therefore molecular characterization is important to allow appropriate genetic counseling. Two genetic subtypes have been described to date: Dent disease 1 is caused by mutations of the CLCN5 gene, coding for the chloride/proton exchanger ClC-5; and Dent disease 2 by mutations of the OCRL gene, coding for the inositol polyphosphate 5-phosphatase OCRL-1. Herein, we review previously reported mutations (n = 192) and their associated phenotype in 377 male patients with Dent disease 1 and describe phenotype and novel (n = 42) and recurrent mutations (n = 24) in a large cohort of 117 Dent disease 1 patients belonging to 90 families. The novel missense and in-frame mutations described were mapped onto a three-dimensional homology model of the ClC-5 protein. This analysis suggests that these mutations affect the dimerization process, helix stability, or transport. The phenotype of our cohort patients supports and extends the phenotype that has been reported in smaller studies.

Characterization of a shorter recombinant polypeptide chain of bone morphogenetic protein 2 on osteoblast behaviour

BACKGROUND Recombinant bone morphogenetic protein two (rhBMP2) has been utilised for a variety of clinical applications in orthopaedic surgery and dental procedures. Despite its widespread use, concerns have been raised regarding its short half-life and transient bioactivity in vivo. Recent investigation aimed at developing rhBMP2 synthesized from a shorter polypeptide chain (108 amino acids) has been undertaken. METHODS The osteopromotive properties of BMP2 were investigated on cell behaviour. Five concentrations of rhBMP2_108 including 10, 50, 100, 200 and 500 ng/ml were compared to a commercially available rhBMP2 (100 ng/ml). Each of the working concentrations of rhBMP2_108 were investigated on MC3T3-E1 osteoblasts for their ability to induce osteoblast recruitment, proliferation and differentiation as assessed by alkaline phosphatase (ALP) staining, alizarin red staining, and real-time PCR for genes encoding ALP, osteocalcin (OCN), collagen-1 (COL-1) and Runx2. RESULTS The results demonstrate that all concentrations of rhBMP2_108 significantly improved cell recruitment and proliferation of osteoblasts at 5 days post seeding. Furthermore, rhBMP2_108 had the most pronounced effects on osteoblast differentiation. It was found that rhBMP2_108 had over a four fold significant increase in ALP activity at seven and 14 days post-seeding and the concentrations ranging from 50 to 200 ng/ml demonstrated the most pronounced effects. Analysis of real-time PCR for genes encoding ALP, OCN, COL-1 and Runx2 further confirmed dose-dependant increases at 14 days post-seeding. Furthermore, alizarin red staining demonstrated a concentration dependant increase in staining at 14 days. CONCLUSION The results from the present study demonstrate that this shorter polypeptide chain of rhBMP2_108 is equally as bioactive as commercially available rhBMP2 for the recruitment of progenitor cells by facilitating their differentiation towards the osteoblast lineage. Future in vivo study are necessary to investigate its bioactivity.

CO2, light and temperature influence senescence and protein degradation in wheat leaf segments

Effects of environmental conditions influencing photosynthesis and photorespiration on senescence and net protein degradation were investigated in segments from the first leaf of young wheat (Triticum aestivum L. cv. Arina) plants. The segments were floated on H2O at 25, 30 or 35°C in continuous light (PAR: 50 or 150 µmol m−2 s−1) in ambient air and in CO2-depleted air. Stromal enzymes, including phosphoglycolate phosphatase, glutamine synthetase, ferredoxin-dependent glutamate synthase, phosphoribulokinase, and the peroxisomal enzyme, glycolate oxidase, were detected by SDS-PAGE followed by immunoblotting with specific antibodies. In general, the net degradation of proteins and chlorophylls was delayed in CO2-depleted air. However, little effect of CO2 on protein degradation was observed at 25°C under the lower level of irradiance. The senescence retardation by the removal of CO2 was most pronounced at 30°C and at the higher irradiance. The stromal enzymes declined in a coordinated manner. Immunoreactive fragments from the degraded polypeptides were in most cases not detectable. However, an insolubilized fragment of glycolate oxidase accumulated in vivo, especially at 25°C in the presence of CO2. Detection of this fragment was minimal after incubation at 30°C and completely absent on blots from segments kept at 35°C. In CO2-depleted air, the fragment was only weakly detectable after incubation at 25°C. The results from these investigations indicate that environmental conditions that influence photosynthesis may interfere with senescence and protein catabolism in wheat leaves.