Research On Mechano-regulation Of Differential Gene Expression And Alternative Splicing In Osteoblasts

Mechanical signals play important roles in regulation of organism. There has beenconcern about this problem. Mechanotransduction is a complex process that serials ofgenes, receptors and pathways are involved in. One cannot underscore enough thecomplexity of mechanotransduction. Therefore, a global analysis of gene expressionsregulated by mechanical stimuli is expected to yield a more extensive view into thebiological roles of mechanical signals and the mechanisms. A systemic understanding ofmechanotransduction is critical for determining how cells proliferate, differentiate andrespond to mechanical stimuli. This study globally investigated the gene expressionprofiling via long serial analysis of gene expression (Long SAGE) in osteoblasts afterexposure to mechanical stretching. The differentially expressed genes were analyzed byfunctional analysis, GO analysis and KEGG pathway analysis. These research resultsprovide a systemic profile of mechanotransduction in osteoblast regulation.Gene expression is regulated by mechanical stress. The possible mechanism of thisprocess is that mechanical stress could regulate alternative splicing of genes. For mosteukaryotic genes, during the transcriptional process pre-mRNAs must undergo severalpost-transcriptional modifications. Alternative splicing is an important kind ofpost-transcriptional modifications. In this study, real time quantitive PCR was used toidentify the splicing factors involved in mechano-regulation of alternative splicing.Function of splicing factors are closely related to phosphorylation. This study detectedthe expression of phosphorylation kinases in response to stretching, and describedphosphorylation pathways participated in mechano-regulation of alternative splicing.The main works and conclusions are included as follows:①Structure of loading model: Osteoblasts were harvested from newborn Wistarrat calvaria by tissue pieces-stick method. After identified by stain, osteoblasts from thesecond to fourth passages were used in the experiment; a cyclic-stretching device wasdesigned to apply cyclic stretching to osteoblast. This device could provide enough cellsfor genome wide analysis.②In response to different kinds of stretching stimulation, the proliferation ofosteoblasts was detected by cell cycle. The result showed that stretching promotedproliferation of osteoblasts. The result of ALP expression showed that stretchinginhibited differentiation. In order to find the effection of stretching on mineralization of osteoblasts, calcium was detected. The result showed that stretching promoted themineralization of osteoblasts.③The expression of several specific genes in osteoblast were detected byRT-PCR. The results demonstrated that mechanical stress could affect specific geneexpression of osteoblasts, consequently affected the proliferation, differentiation andmineralization of osteoblasts.④Controlled and stretched libraries were constructed by Long SAGE.Compared to the controlled library, in stretched library100tags were significantlyup-regulated and72tags were significantly down-regulated on the base of statisticalanalysis. These results suggested that mechanical stress could efficiently regulateexpression of genes.⑤In order to validate the two Long SAGE libraries, six differentially expressedgenes were randomly selected. The expression levels of genes were tested by real-timePCR. The comparison of the results of real-time PCR and Long SAGE indicated thatthe expression of genes in real-time PCR was generally in accordance with the LongSAGE data. Long SAGE data were true and reliable.⑥Differentially expressed genes from Long SAGE data were analyzed bybioinformation method. The result of function analysis showed that these differentialgenes played roles in biosynthesis of protein, signal transduction, metabolism, ionbinding, development, apoptosis, cell adhension, cytoskeleton, proliferation and cellmovement. GO analysis showed that these differential genes belonged to different GOcategories. Mechanical stress affected physiology and behavior of osteoblasts byregulating gene expression. KEGG pathway analysis showed that these differentialgenes involved in three different signal pathways, including Ribosome pathway, Focaladhesion pathway and ECM-receptor interaction pathway.⑦This study globally investigated the gene expression profiling via Long SAGEin osteoblasts after exposure to mechanical stretching. Upon sensation of themechanical cues, several membrane-bound receptors such as integrins, Ca2+channels,DAG, Wnt, BMP-6/TGF-β receptors are activated, resulting in the activation of severalpathways including ECM-integrin, Actin, ERK1/2, BMP-6/TGF-β, Wnt,Ca2+-regulation and NO regulation pathway. And then mechanical stretching signalsare propagated through cytoplasm into the nucleus eventually to regulate thetranscription of genes associated with pro-mRNA splicing, ECM components,cytoskeleton reorganization, osteoblast proliferation, differentiation and apoptosis. ⑧This study identified splicing factors participated in mechano-regulation ofalternative splicing. SR proteins, including ASF/SF2and SC35, hnRNP protein, such ashnRNP A1, were detected by real time quantitive PCR. Results showed that these twokinds of splicing factors could be regulated by mechanical stress. So it could besupposed that splicing isoform expression was related to splicing factors regulated bymechanical stress. Phosphorylation of the RS domains in SR proteins modulates theirsub-cellular localization and activation, which decide the selection of alternativesplicing sites. Various kinases react on this process, including SRPK1, SRPK2, PI3K,CLK1and PP1. These kinases increased significantly in response to stretching. Basedon these results, the phosphorylation model of alternative splicing mechano-regulationwas proposed.