Ischemia/reperfusion reduces transcription factor Sp1-mediated cystathionine beta-synthase expression in the kidney

Abstract

Cystathionine beta-synthase (CBS) is a key enzyme that catalyzes the rate-limiting step for homocysteine (Hcy) metabolism via the trans-sulfuration pathway and is also responsible for the production of H(2)S through the desulfhydration reaction. Our recent studies demonstrate that renal ischemia/reperfusion decreased the CBS activity leading to Hcy accumulation and H(2)S reduction in the kidney, which in turn contributed to kidney injury. Both Hcy and H(2)S play important roles in physiological and pathological processes. In this study we investigated the molecular mechanism by which CBS activity was regulated in the kidney. The left kidney of Sprague-Dawley rat was subjected to 45 min of ischemia followed by 6 h of reperfusion. Ischemia/reperfusion caused a significant decrease in CBS mRNA and protein levels in the kidney. As a consequence, there was a marked reduction in the CBS enzyme activity. Transfection of kidney proximal tubular cells with transcription factor (Sp1) small interfering RNA caused a marked reduction in CBS mRNA, indicating a pivotal role for Sp1 in regulating CBS expression in kidney cells. Electrophoretic mobility shift assay and chromatin immunoprecipitation assay detected a lower Sp1 activity in kidneys subjected to ischemia/reperfusion as compared with that in a sham-operated group. ERK-mediated phosphorylation of Sp1 was responsible for a decreased transcriptional activity of Sp1 in the kidney upon ischemia/reperfusion. These results suggest that reduced kidney CBS gene expression during ischemia/reperfusion is mediated via a decrease in Sp1 transcriptional activity. Regulation of CBS-mediated Hcy and H(2)S homeostasis may offer a renal protective effect against ischemia/reperfusion injury.

FIGURE 1.

CBS mRNA and protein expression in the kidney. A, the left kidney was subjected to sham operation (Sham) or ischemia/reperfusion (I/R). CBS mRNA in the kidney tissue was determined by real time PCR assay. Crossing threshold values were normalized to GAPDH expression. B, CBS protein in kidney was determined by Western immunoblotting analysis. C, human proximal tubular epithelial cells (HK2) were subjected to ischemic conditions for 2 h followed by incubation in the maintenance medium for 6 h (I/R) as described under “Experimental Procedures.” CBS mRNA in control cells and in ischemia/reperfusion cells was determined by real time PCR assay. The results are expressed as the means ± S.E. from six separate experiments, each performed in duplicate. *, p < 0.05 when compared with the value obtained from sham-operated group (A and B) or when compared with the value obtained from control cells (C).

FIGURE 2.

Effect of ischemia/reperfusion on Sp1 activation in the kidney. The left kidney was subjected to sham operation (Sham) or ischemia/reperfusion (I/R). A, the Sp1 protein level in the kidney tissue was determined by Western immunoblotting analysis. B, the DNA binding activity of Sp1 in the kidney tissue was determined by EMSA. The area of the Sp1-DNA bands in the EMSA gel was cut out according to the autoradiograph image, and the radioactivity corresponding to each band was measured using a liquid scintillation counter. The average radioactivity detected in the sham-operated group was 61,259 cpm and was expressed as 100%. C, nuclear proteins were incubated with anti-Sp1 antibodies (Sp1 Ab) or anti-phosphoserine antibodies (p-Ser Ab) for supershift assays. D, a schematic diagram shows the CBS promoter 5′-untranslated region. The bold letters indicate the potential binding sequences for Sp1. The transcription starting site of the rat CBS gene is indicated by an asterisk. ChIP assay was conducted in the kidney tissue. The expression of CBS promoter region associated with Sp1 was determined by real time PCR assay. As an internal control, an aliquot of sheared chromatin unincubated with antibodies was prepared (mock immunoprecipitation) to normalize the amount of total DNA subjected to ChIP assay. The results are expressed as the means ± S.E. from four separate experiments, each performed in duplicate. *, p < 0.05 when compared with the value obtained from the sham-operated group.

FIGURE 3.

Effect of Sp1 siRNA transfection on CBS expression in proximal tubular cells. Human proximal tubular cells were transiently transfected with Sp1 siRNA or with a scramble siRNA (Negative control). Sp1 mRNA expression (A) and CBS mRNA expression (C) were determined by a real time PCR assay. Crossing threshold values were normalized to GAPDH expression. Sp1 protein expression (B) and CBS protein expression (D) were determined by Western immunoblotting analysis. The mRNA or protein expression in negative control cells (transfected with scrambled siRNA) was expressed as 100%. The results are expressed as the means ± S.E. from six separate experiments, each performed in duplicate. *, p < 0.05 when compared with the value obtained from the negative control.

FIGURE 4.

Effect of ischemia/reperfusion on phosphorylation of Sp1 in the kidney. The left kidney was subjected to sham operation (Sham) or ischemia/reperfusion (I/R). A, Sp1 protein in the kidney tissue was immunoprecipitated (IP) using anti-Sp1 antibodies followed by Western immunoblotting (IB) analysis using anti-phosphoserine antibodies to detect serine-phosphorylated Sp1 protein. The total Sp1 protein in the immunoprecipitate was determined by Western immunoblotting analysis using polyclonal anti-Sp1 antibodies. Phosphorylated Sp1 protein level was normalized to the total Sp1 detected in the immunoprecipitate. B, dephosphorylation of Sp1 increased Sp1-DNA binding activity in ischemia/reperfusion. Nuclear proteins from ischemia/reperfused kidney tissue were incubated with or without calf intestinal phosphatase (CIP, 0.2 milliunits/μg of nuclear proteins). EMSA was performed to determine the Sp1-DNA binding activity. The area of the Sp1-DNA bands in the EMSA gel was cut out according to the autoradiograph image, and the radioactivity corresponding to each band was measured using a liquid scintillation counter. The average radioactivity detected in the sham-operated group was expressed as 100%. The results are expressed as the means ± S.E. from four separate experiments, each performed in duplicate. *, p < 0.05 when compared with the value obtained from the sham-operated group. #, p < 0.05 when compared with the value obtained from the ischemia/reperfusion group.

FIGURE 5.

Effect of ischemia/reperfusion on ERK activation in the kidney. The left kidney was subjected to sham operation (Sham) or ischemia/reperfusion (I/R). Phosphorylated ERK1/2 protein (p-ERK) and total ERK1/2 protein in the kidney were determined by Western immunoblotting analysis. The results are expressed as the means ± S.E. from four separate experiments, each performed in duplicate. *, p < 0.05 when compared with the value obtained from the sham-operated group.

FIGURE 6.

Effect of ERK inhibitor on Sp1 activation and CBS expression in proximal tubular cells. A, human proximal tubular epithelial cells (HK2) were pretreated with 10 μm ERK inhibitor, PD98059, for 30 min and then subjected to ischemic condition for 2 h followed by incubation in the maintenance medium for 6 h (I/R) in the presence of PD98059 (I/R+PD). Sp1-DNA binding activity was determined by EMSA. The area of the Sp1-DNA bands in the EMSA gel was cut out according to the autoradiograph image, and the radioactivity corresponding to each band was measured using a liquid scintillation counter. The average radioactivity detected in the sham-operated group was expressed as 100%. B, CBS mRNA expression in ischemia/reperfused cells with or without PD98059 treatment was determined by real time PCR assay. Crossing threshold values were normalized to GAPDH expression. The results are expressed as the means ± S.E. from four separate experiments, each performed in duplicate. *, p < 0.05 when compared with the value obtained from control cells. #, p < 0.05 when compared with the value obtained from cells subjected to ischemia/reperfusion.

FIGURE 7.

Effect of ischemia/reperfusion on CBS activity, Hcy, and H₂S levels in the kidney. The left kidney was subjected to sham operation (Sham) or ischemia/reperfusion (I/R). A, the CBS activity in the kidney tissue was determined. B and C, the levels of Hcy (B) and H₂S (C) in the kidney tissue were measured. The results are expressed as the means ± S.E. from six separate experiments, each performed in duplicate. *, p < 0.05 when compared with the value obtained from the sham-operated group.