Cystathionine β‐synthase is coordinately regulated with proliferation through a redox‐sensitive mechanism in cultured human cells and Saccharomyces cerevisiae

Abstract

Cystathionine beta-synthase (CBS) catalyzes the condensation of serine with homocysteine to form cystathionine and occupies a crucial regulatory position between the methionine cycle and the biosynthesis of cysteine by transsulfuration. Analysis of CBS activity under a variety of growth conditions indicated that CBS is coordinately regulated with proliferation in both yeast and human cells. In batch cultures of Saccharomyces cerevisiae, maximal CBS activities were observed in the exponential phase of cells grown on glucose, while growth-arrested cultures or those growing non-fermentatively on ethanol or glycerol had approximately 3-fold less activity. CBS activity assays and Western blotting indicated that growth-specific regulation of CBS is evolutionarily conserved in a range of human cell lines. CBS activity was found to be maximal during proliferation and was reduced two- to five-fold when cells became quiescent at confluence. In cultured HepG2 cells, the human CBS gene is induced by serum and basic fibroblast growth factor and is downregulated, but not abolished, by contact inhibition, serum-starvation, nutrient depletion, or the induction of differentiation. Consequently, for certain cell types, CBS may represent a novel marker of both differentiation and proliferation. The intracellular level of the CBS regulator compound, S-adenosylmethionine, was found to reflect the proliferation status of both yeast and human cells, and as such, constitutes an additional mechanism for proliferation-specific regulation of human CBS. Our data indicates that screening compounds for the ability to affect transsulfuration in cultured cell models must take proliferation status into account to avoid masking regulatory interactions that may be of significance in vivo.

The Dominant Role of Sp1 in Regulating the Cystathionine β-Synthase –1a and –1b Promoters Facilitates Potential Tissue-specific Regulation by Kruppel-like Factors

“…Media and Mammalian Cell Culture-HepG2 hepatocellular carcinoma cells were obtained from the American Type Culture Collection (Manassas, VA) and were cultured as described previously (10). Drosophila SL2 (Schneider) cells were a generous gift from Dr. Joan Hooper (Department of Cellular and Structural Biology, University of Colorado Health Sciences Center).…”

“…The LKLF mammalian expression construct pBKCMVwtLKLF was a generous gift from Dr. Jerry Lingrel (University of Cincinnati College of Medicine, OH) and the KLF8 and BKLF expression constructs pMT2.BKLF, pMT2.KLF8, and pPac.BKLF were kindly provided by Dr. Merlin Crossley (University of Sydney, New South Wales, Australia). Transient transfections and subsequent luciferase reporter assays in actively proliferating cells were performed as described previously (10).…”

“…2A) shows one candidate Sp1 binding site that overlaps with a binding site for the zinc-finger transcription factor early growth-response protein (Egr1), one CCAAT box, and putative binding sites for Ap2, MZF1, IK2, and Gfi1. Ap2 is unlikely to contribute to the promoter activity of this construct, because it is not expressed in HepG2 cells (20) and has previously been shown to downregulate CBS when heterologously expressed in these cells (10). The tissue distribution of Gfi1, IK2, and MZF1 (21) (22-24) effectively precludes any role for these transcription factors in the regulation of CBS in tissues such as liver, kidney, pancreas, and brain, which are the main sites of transsulfuration in humans (8).…”

“…Numbers in parentheses define the amount of base pairs of Ϫ1b proximal sequence. Transient transfections into HepG2 cells and subsequent luciferase-based promoter reporter assays were performed as described previously (10). All of the promoter constructs used are derivatives of our previously described CBS Ϫ1b promoter construct pCBS47 (6) and contain a common 3Ј-end.…”

“…The temporal and spatial expression patterns of CBS appear to be developmentally regulated (8 -10). Recently, we have shown that both human and yeast CBS are coordinately regulated with proliferation and that the human CBS Ϫ1b promoter is serum-and fibroblast growth factor-responsive and is downregulated by growth arrest due to nutrient depletion or the action of differentiation-inducing reagents (10,11). Another group has recently investigated the CBS Ϫ1b promoter and defined the minimal promoter region as a 125-bp region of sequence proximal to exon Ϫ1b.…”