Sd Rats Into The Interaction Of Bmp And Pka / Pkc Signaling Pathways In Bone Cells

Growth factors are polypeptides that bind to corresponding cell membrane receptors to stimulate or inhibit certain functions within cells. There are two different types of BMP specific serine/threonine kinase receptors on some types of cell membrane: type I and type II. Type II receptor kinase is constitutively activate. Upon ligand binding, type II receptor activates type I receptor kinases through phosphorylation of the juxtamembrane domain of type I receptors. Type I receptor kinases then activate intracellular substrate, the central signal messengers being Smad proteins. Receptor-regulated Smads (R-Smads) directly interact with type I receptors and become phosphorylated. R-Smads then form heterocomplexs with Common Smads (C-Smads) and migrate into the nucleus, where they regulate transcription of target genes. PKAs and PK.Cs are two different families of protein kinases in mammal cells, regulating much biological behaviors of the cells such as substance metabolism, expression of genes, proliferation and differentiation. The present study is focused on the physiological effects of PKAs and PK.Cs, and their interaction with BMPs in SD rat osteoblasts, including three main parts:1 Culture model of SD rat ostcoblast cells in vitro. The primary culture of osteoblasts was conducted as reports described in mineralization inducing medium. Subculture and purification of the cells were performed by trypsinization according to different anchorage potential of osteoblasts from other types of cells. The cultured osteoblasts were fusiform polygonal starlike with long and thin processes. The doubling time was 90.6 hours, and anchorage ratio to the Petri dish floor was 90% 18 hours after seeding. Positive staining of ALP and type I collagen was found in almost all anchoring cells. By chinalizarin staining calcium nodules were found in the centre of concentrated cells. DMSO at Low concentration(below 1%) showed no significant effection on osteoblasts. The osteoblasts had been cultured as description above and synchronized before following experiments were to be conducted.2 Effect of PKAs on the SD rat osteoblast cells(l)The cells were treated with cAMP (ImM). PKI (Img/L). rhBMP-2 (200ug/L), PKl-rhBMP-2 (PKI pretreating for 1 hour) respectively in different groups. The results showed that there was no significant difference in proliferation among the groups (p>0.05). But cAMP heightened ALP activity after treating for 60 minutes, where as PKI reduced ALP activaty (p<0.05). More minerialized nodules in Group cAMP was found than in Group PKI (p<0.01).(2)Expression of PKA in SD rat osteoblast cells: The cells were treated with cAMP. PKI. rhBMP-2 respectively for 30 minutes. Then cPKA.,and cPKA, were detected through immunocytochemistry. The cells in all groups were positive for cPKA, and cPKA,t, but those in Group PKI were the weakest and the strongest were in Group cAMP studied by image analysis.(3)Effect of BMP on PKA activity: The cells were treated with rhBMP-2 or PKl-rhBMP-2 (PKI pretreating for 30 minutes) for 10. 30. 60 minutes respectively. Then PKA activity was measured with 32P incorperation test. rhBMP-2 increased PKA activity after treating for 30 minutes (p<0.05). PKA activity was decreased after PKI pretreated for 30 minutes, whereas rhBMP-2 elevated the depressed PKA activty in a time-dependent manner(p<0.05).(4)Pilot study on BMP-PKA cross talk: The cells were treated with rhBMP-2 for 10. 20, 30 minutes respectively for cAMP test and 30. 60 minutes respectively for PKA mRNA test. Then the amount of cAMP was examined through RIA. No difference was found between experimental group and control group (p>0.05). PKA mRNA was detected through RT-PCR. and within 60 minutes, there was no significant effect of rhBMP-2 on rat cells PKA mRNA level (p>0.05).3 Effect of PKCs on the SD rat osteoblast cells(l)The cells were treated with PMA (l0mM). PC (l00uM), rhBMP-2 (200ug/L), PC-rhBMP-2 (PC pretreating for 30 minutes) in different groups. There \\as no significant...