The Effects of Sodium Butyrate on Transcription Are Mediated through Activation of a Protein Phosphatase

Abstract

In this study we have investigated the molecular mechanism by which sodium butyrate modulates gene expression when added to cultured cells. As a model system we used hepatoma tissue culture cells in which sodium butyrate treatment increases histone H1°mRNA level and decreases c-myc mRNA level. Because we observed that stimulation of histone H1°gene expression could take place in the absence of protein neosynthesis, we hypothesized that sodium butyrate induced a posttranslational modification of a factor involved in the transcription process. Using different types of well known kinase and phosphatase inhibitors, we studied the implication of kinase or phosphatase activity in this pathway. Interestingly, cell treatment with potent serine-threonine-phosphatase inhibitors, calyculin A or okadaic acid, prevented the regulation of both histone H1°and c-myc gene expressions by sodium butyrate. On the other hand, the tyrosine phosphatase inhibitor, vanadate, or the protein kinase C inhibitor, staurosporine, did not significantly modify sodium butyrate effects. Using protein phosphatase 1 and 2A for in vitro assays, we found a 45% increase of phosphatase activity after cell treatment by sodium butyrate, possibly due to a protein phosphatase 1-type protein phosphatase. These data strongly suggest that signaling pathway(s) triggered by sodium butyrate to modulate gene expression involve(s) a serine-threonine-phosphatase activity.

Quantitative analysis of histone H1° protein synthesis in HTC cells

“…Cell growth arrest or differentiation induced by either serum deprivation or drugs, including sodium butyrate, hexamethylene bis-acetamide (HMBA) and dimethyl sulfoxide, were shown to increase H18 mRNA and protein levels in most mammalian cells in culture. Sodium butyrate induction led to the rapid accumulation of a 2.2-kb H18 mRNA in HeLa cells [2], B16 murine melanoma cells [3] and rat hepatoma tissue culture (HTC) cells used in this study [4]. Unlike HeLa and B16 cells which are stimulated to differentiate, HTC cells are progressively blocked at the early G1 phase of the cell cycle under sodium butyrate treatment [5,6].…”

“…RNAs (30 mg) were fractionated on 0.8% agarose gels containing 2.2 m formaldehyde, transferred onto Genescreen Plus nylon membranes and hybridized with a rat histone H18 coding fragment [4] previously labeled with the random megaprime system (Amersham Corp.). After hybridization, the filters were washed at 42 8C in 0.2 £ NaCl/Cit, 0.1% SDS and then exposed for autoradiography to XAR5 Kodak films for 2 days at 270 8C with an intensifying screen.…”

“…Because this effect is reversible upon removal of the inducer, a 24-h sodium butyrate treatment can therefore be used for HTC cell synchronization [5]. Experiments performed on MEL, B16 and HTC cells showed that H18 mRNA accumulation resulted from an increase of H18 transcription rate [3,4,7], which did not require protein synthesis when stimulated by either HMBA [7] [4]. In the MEL cell line, H18 mRNA preferentially accumulates in mid or late S phase, even when cells have been pulse-induced by a 2-h treatment with HMBA [12,13].…”

“…Because this effect is reversible upon removal of the inducer, a 24-h sodium butyrate treatment can therefore be used for HTC cell synchronization [5]. Experiments performed on MEL, B16 and HTC cells showed that H18 mRNA accumulation resulted from an increase of H18 transcription rate [3,4,7], which did not require protein synthesis when stimulated by either HMBA [7] or sodium butyrate [4]. Although the H18 promoter has been isolated from mouse [8], Xenopus laevis [9], human [10] and rat [11], little is known about factors involved in the regulation of the gene.…”

“…Although the H18 promoter has been isolated from mouse [8], Xenopus laevis [9], human [10] and rat [11], little is known about factors involved in the regulation of the gene. We have recently shown that sodium butyrate activates H18 gene expression in HTC cells by modulating a serine/threonine phosphatase activity [4]. In the MEL cell line, H18 mRNA preferentially accumulates in mid or late S phase, even when cells have been pulse-induced by a 2-h treatment with HMBA [12,13].…”