Screening of genes those are related to dynamic transition of myofiber of vascular smooth muscle cell upon phenotypic modulation.

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Vascular smooth muscle cell (VSMC) shows two typical phenotype, contraction-type and synthetic-type upon various situations. We developed model system of phenotypic modulation by using cultured VSMC, P53LMAC01. Upon addition of PDGF, the VSMC changed to synthetic-type phenotype, and it changed to typical contraction-type phenotype in the presence of sodium butyrate (Na-B), which induce growth suppression by inhibiting acetylation of DNA. We isolated mRNA from these typical two types of cell and constructed subtraction cDNA library. We sequenced inserts of the library, and screened 230 clones from PDGF-specific library, and 160 clones from NaB-specific library. Further inserts of each clone were spotted to nylon membrane and examined by putative Northern hybridization to confirm the specificity in each types of cell. Four sets of membrane was hybridized with four sets of probes, two subtracted cDNAs and two original cDNAs from PDGF-and NaB-treated cells. As a result, 15 clones from PDGF-specific library and 19 clones from NaB-specific library were confirmed in the specificity of the expression. To quantify the expression level of these clones in PDGF-and NaB-treated cell, we made primer sets for each clone and examined with real time PCR. Clones showing 2-fold difference of expression among these two types of cell were S 100A4, S100A6, α-actin and others. We isolated the full-length cDNA of S 100A4 and S 100A6 by RT-PCR, and expressed these genes in bacteria. Using these gene products we affinity purified three proteins that interact with S 1004 and S100A6 proteins from the VSMC.