Sp6 downregulation of follistatin gene expression in ameloblasts

Abstract

: Sp6 is a member of the Sp family of transcription factors that regulate a wide range of cellular functions, such as cell growth and differentiation. Sp6, also called epiprofin, is specifically expressed in tooth germ, limb bud, and hair follicle, but there is little information on its function.To investigate the possible role of Sp6 in tooth development, first we established an Sp6-overproducing clone, CHA9, and analyzed the features of the cell, including cell proliferation and gene expression. The parental cells of CHA9 are the ameloblast-lineage G5 cells that we previously established from rat dental epithelia of lower incisor. Sp6 overproduction accelerated cell proliferation and induced the expression of ameloblastin mRNA, a marker of ameloblast differentiation. Second, we performed genome-wide screening of Sp6 target genes by microarray analysis. Out of a total 20,450 genes, 448 genes were up-regulated and 500 genes were down-regulated by Sp6. We found the expression of follistatin, a BMP antagonist, to be 22.4-fold lower in CHA9 than in control cells. Transfection of the Sp6-antisense construct into CHA9 cells restored follistatin expression back to equivalent levels seen in control cells, indicating that Sp6 regulates follistatin gene expression in ameloblasts.Our findings demonstrate that the follistatin gene is one of the Sp6 target genes in ameloblasts and suggest that Sp6 promotes amelogenesis through inhibition of follistatin gene expression. J. Med. Invest. 55 : 87-98, February, 2008

Sp6 regulation of <i>Rock1 </i>promoter activity in dental epithelial cells

“…Immunohistochemical analyses demonstrated that Sp6 and ROCK1 express sequentially (3,9). Cytosolic ROCK1 were detected at low level in basal epithelial cells, inner enamel epithelium cells, and presecretory ameloblasts (9).…”

“…The PCR fragments and the luciferase reporter vector pGL3-basic were separately digested with SacI and HindIII before ligation, and reporter constructs were sequenced. Sp6 expression vector was described previously (3). Sp1 expression vector was constructed using pCIneo mammalian expression vector (Promega).…”

“…Sp1 expression vector was constructed using pCIneo mammalian expression vector (Promega). In brief, the entire rat Sp1 coding region was generated by RT-PCR using PrimeSTAR GXL DNA Polymerase (Takara) from total RNA of C9 cells (3). FLAG-tag was added at the N-terminus of Sp1 cDNA in the forward primer.…”

BMP2-induced gene profiling in dental epithelial cell line

“…Lysates were separated by centrifugation at 20,000!g for 15 min and supernatants were collected in a microtube. Measurement of protein contents and Western blot analysis were carried out as described previously (17). Activated Smad1/5/8 was detected using antiphospho -Smad1/5/8 (1 : 1,000 ; #9511, Cell Signaling, Danvers, MA, USA).…”

“…The arrays were scanned and analyzed. Differential expression of the genes was determined as previously reported (17).…”