SRY-related HMG box 9 regulates the expression of Col4a2 through transactivating its enhancer element in mesangial cells - PubMed (original) (raw)
SRY-related HMG box 9 regulates the expression of Col4a2 through transactivating its enhancer element in mesangial cells
Eriko Sumi et al. Am J Pathol. 2007 Jun.
Abstract
Accumulation of alpha1(IV) and alpha2(IV) collagen is one of the characteristic pathological changes in glomerulosclerosis. Although the Col4a2 gene is known to have a 0.3-kb critical enhancer element with the GAACAAT motif, which transcription factor binds and transactivates this motif has not been identified. In this study, we found that SRY-related HMG box 9 (SOX9) was bound to the GAACAAT motif in the Col4a2 enhancer in vitro and in vivo in mesangial cells. SOX9 strongly activated this enhancer when cotransfected with Col4a2 enhancer-promoter construct in mesangial cells and Swiss/3T3 cells. Mutation in the GAACAAT motif eliminated the activation by SOX9. Furthermore, transforming growth factor-beta (TGF-beta) treatment induced the expression of SOX9 and Col4a2, and a small interfering RNA against SOX9 reduced Col4a2 expression induced by TGF-beta treatment in mesangial cells. In vivo, we found that the expression of SOX9 was dramatically increased along with the expression of TGF-beta and Col4a2 in mouse nephrotoxic nephritis. These results indicate that SOX9 is essential for Col4a2 expression in mesangial cells and might be involved in the accumulation of alpha2(IV) collagen in experimental nephritis.
Figures
Figure 1
A: TGF-β increased SOX9 and α2(IV) collagen proteins in mesangial cells. Mouse mesangial cells were cultured for the indicated periods of time in the presence or absence of 1 ng/ml TGF-β. Total cell lysates were examined by immunoblotting analysis using anti-SOX9 and anti-type IV collagen antibodies. An actin immunoblot was used to control for equal loading. The figure presents data from one of three experiments that produced similar results. B and C: Optical densitometry of SOX9 and α2(IV) collagen in immunoblotting. The values of SOX9 and α2(IV) collagen were normalized for that of actin and compared with the values of 0 hours. The average value of three independent experiments is shown. *P < 0.05 compared with control.
Figure 2
AACAAT of the 0.3-kb enhancer element is critical for activation in mesangial cells. A: Genomic map of the mouse Col4a1 and Col4a2 genes with exons of Col4a1 and Col4a2 genes (black boxes) and the orientation of transcription initiation sites are shown. Scale bar indicates the distance (bp) of fragments relative to Col4a1 gene transcription start site. Enhancer and promoter fragments used during construction of plasmids are indicated as gray box and open box, respectively. Arrowheads show primers used in ChIP assay. B: The sequences of these promoter and enhancer elements were shown. The number indicates the distance (bp) of fragments relative to Col4a1 gene transcription start site. The left and right brackets in the sequence indicate the end points of deletions. The underlined sequences are former and latter GAACAAT motif. C: Schematic diagram of promoter and wild-type, deleted, or mutated enhancer elements indicated by the positions of the first and last nucleotides relative to the transcription start site of Col4a1 gene (left). Col4a2 promoter is displayed as a white box, and enhancer is displayed as a gray box. The wild-type critical element, AACAAT, is displayed as a black box, and the mutated one, AACGCT, is displayed as a dark gray box. Activities of the enhancer-promoter reporter constructs in mesangial cells are shown. Mesangial cells were transfected with 1 μg of reporter construct with 100 ng of firefly luciferase under the control of cytomegalovirus promoter as an internal control. The average values of three independent experiments in triplicate are shown as fold increase compared with that of no enhancer. *P < 0.05 compared with control.
Figure 3
SOX9 bound to the Col4a2 enhancer element through GAACAAT motif in vitro. A: Electrophoretic mobility shift assay was performed using the Col4a2 enhancer probe with in vitro synthesized flagged SOX9. SOX9 bound to the Col4a2 enhancer probe (lanes 1 and 2), and the DNA-protein complex was supershifted by adding antibodies against Flag (lane 4). An arrow indicates supershifted SOX9-DNA complexes. B: Electrophoretic mobility shift assay was performed using in vitro synthesized SOX9 with unlabeled wild-type or mutated probes as competitors. SOX9-DNA complex was competed out by an excess amount of wild-type probes but not by mutated probes in the GAACAAT motif.
Figure 4
SOX9 interacts with the Col4a2 gene enhancer in vivo. A and B: Chromatin prepared from mesangial cells treated with TGF-β or vehicle was immunoprecipitated with IgG or SOX9 antibody as indicated. PCR was done with oligonucleotide pairs for two regions of the Col4a1 gene, one corresponding to the enhancer region (−4259 to −4100) containing the GAACAAT (enhancer with GAACAAT; A) and the other one corresponding to an upstream region (−7252 to −7058) (negative control; B). Ten microliters of sonicated and precleared chromatin was used as input DNA. MW, molecular weight.
Figure 5
SOX9 activated the enhancer-promoter reporter constructs. A: Activation of the enhancer-promoter reporter construct by SOX9 in mesangial cells. Mesangial cells were transfected with 1 μg of reporter construct with 100 ng of firefly luciferase under the control of cytomegalovirus promoter as an internal control. The average values of three independent experiments in triplicate are shown as fold increase compared with that of no enhancer. B: Activation of wild-type or mutant type enhancer-promoter reporter constructs by SOX9 in Swiss/3T3 cells. *P < 0.05 compared with control.
Figure 6
Involvement of SOX9 in Col4a2 up-regulation in mesangial cells. A: SOX9 mRNA and protein was reduced by transfection of siRNA against SOX9 in cells treated or untreated with TGF-β. Gene expression of SOX9 was examined by quantitative RT-PCR using mRNA of mesangial cells transfected with siRNA against SOX9 or control RNAi (a). An immunoblot analysis of mesangial cells transfected with siRNA against SOX9 or control RNAi, treated with TGF-β or nontreated (b). Optical densitometry of SOX9 in immunoblotting (c). The average value of three independent experiments was shown. The results were presented as the fold increase or decrease compared with the values of cells transfected with control RNAi and nontreated cells. B: Col4a2 mRNA expression was suppressed by transfection of siRNA against SOX9 (a). Optical densitometry of α2(IV) collagen in immunoblotting (b). Col4a1 mRNA expression was also suppressed by transfection of siRNA against SOX9 (c). The average value of three independent experiments was shown. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. *P < 0.05 compared with control.
Figure 7
Up-regulation of SOX9 in NTN. A: Mice were sacrificed before induction of NTN and at days 7 and 14 after induction of NTN. Gene expression of SOX9 was examined by quantitative RT-PCR using whole-kidney mRNA. The results were presented as the fold increase compared with the values obtained before the induction of NTN (day 0). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. B: Immunohistological analysis of SOX9 in NTN. Note that nuclear staining of SOX9 was seen in the glomeruli at day 7 (b), although SOX9 was hardly expressed in the glomeruli before induction (a). *P < 0.05 compared with control.
Figure 8
Expression of TGF-β, Col4a1, and Col4a2 in NTN. Gene expression of TGF-β (A) and Col4a1 and Col4a2 (B) was examined by quantitative RT-PCR using whole-kidney mRNA. The results were presented as the fold increase compared with the values obtained before the induction of NTN (day 0). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. *P < 0.05 compared with control. C: Immunohistochemical findings of TGF-β (a and b) and α1 and α2(IV) collagen (c and d) at control (a and c) and day 7 (b and d). TGF-β was up-regulated in the glomeruli at day 7 (b), although it was hardly seen before induction (a). Accumulation of α1 and α2(IV) collagen was also observed at day 7 (d) compared with before induction (c). White scale bar = 100 μm. Original magnifications, ×400.
Figure 9
Light microscopy. Periodic acid-Schiff methenamine-stained paraffin-embedded sections of mouse kidney before induction of NTN (a) and at day 7 (b) are shown. Increased mesangial cell number and extracellular matrix were seen in large glomeruli at day 7. White scale bar = 100 μm. Original magnifications, ×400.
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