Critical role of the HMGI(Y) proteins in adipocytic cell growth and differentiation - PubMed (original) (raw)

. 2001 Apr;21(7):2485-95.

doi: 10.1128/MCB.21.7.2485-2495.2001.

G M Pierantoni, S Scala, S Battista, M Fedele, A Stella, M C De Biasio, G Chiappetta, V Fidanza, G Condorelli, M Santoro, C M Croce, G Viglietto, A Fusco

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Critical role of the HMGI(Y) proteins in adipocytic cell growth and differentiation

R M Melillo et al. Mol Cell Biol. 2001 Apr.

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Abstract

The high-mobility group I (HMGI) nonhistone chromosomal proteins HMGI(Y) and HMGI-C have been implicated in defining chromatin structure and in regulating the transcription of several genes. These proteins have been implicated in adipocyte homeostasis: a severe deficiency of fat tissue is found in mice with targeted disruption of the HMGI-C locus, and lipomagenesis in humans is frequently associated with somatic mutations of HMGI genes. The aim of this study was to examine the role of HMGI(Y) proteins in adipocytic cell growth and differentiation. First, we found that differentiation of the preadipocytic 3T3-L1 cell line caused early induction of HMGI(Y) gene expression. Suppression of HMGI(Y) expression by antisense technology dramatically increased the growth rate and impaired adipocytic differentiation in these cells. The process of adipogenic differentiation involves the interplay of several transcription factors, among which is the CCAAT/enhancer-binding protein (C/EBP) family of proteins. These factors are required for the transcriptional activation of adipocyte-specific genes. We also tested the hypothesis that HMGI(Y) might participate in transcriptional control of adipocyte-specific promoters. We found that HMGI(Y) proteins bind C/EBPbeta in vivo and in vitro. Furthermore, we show that HMGI(Y) strongly potentiates the capacity of C/EBPbeta to transactivate the leptin promoter, an adipose-specific promoter. Taken together, these results indicate that the HMGI(Y) proteins play a critical role in adipocytic cell growth and differentiation.

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Figures

FIG. 1

FIG. 1

(A) HMGI(Y) induction during NIH 3T3-L1 preadipocyte differentiation. Total RNA (20 μg/lane) extracted from proliferating and differentiated 3T3-L1 cells was hybridized with the HMGI(Y) cDNA and then with a rat GAPDH probe as a control for RNA loading. RNA was extracted from undifferentiated proliferating cells (P) at time zero and at 6 h, 1 day, and 4 days of differentiation, as indicated. (B) Nuclear proteins extracted from normal and induced 3T3-L1 cells were separated (20 μg/lane) by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Western blots were incubated first with antibodies specific for HMGI(Y) proteins and then with horseradish peroxidase-conjugated secondary antibodies; the immunocomplexes were detected by enhanced chemiluminescence. As a control for equal loading, the blotted proteins were stained with Ponceau Red. Moreover, the same Western blots were incubated with antibodies to the ubiquitous γ-tubulin protein. Proteins were extracted from the same cells as in panel A.

FIG. 2

FIG. 2

(A) HMGI(Y) sense and antisense constructs were generated as described in Materials and Methods. The untranslated region, DNA-binding domain, and acidic domain of the HMGI(Y) protein are indicated. (B) RT-PCR analysis of pHMGI(Y)s expression in 3T3-L1-HMGI(Y)s cells. The sources of RNAs are the following: lane 1, PCR on the pCMV-HMGI(Y)s plasmid (positive control); lanes 2, 3, and 4, 3T3-L1-HMGI(Y)s (cell clones 1, 2, and 3); lane 5, 3T3-L1 pCMVneo clone 1. (B′) RT-PCR analysis of pHMGI(Y)as expression in 3T3-L1-HMGI(Y)as cells. The sources of RNAs are the following: lane 1, PCR on a pCMV-HMGI(Y)as plasmid (positive control); lanes 2, 3, and 4, 3T3-L1-HMGI(Y)as (cell clones 1, 2 and 3); lane 5, 3T3-L1 pCMVneo clone 1. All cDNAs were coamplified with GAPDH as an internal control. Bands of comparable intensity, obtained by the GAPDH sequence-specific primers, suggest comparable amplification of all samples. (C) 3T3-L1, 3T3-L1-HMGI(Y)as, 3T3-L1-HMGI(Y)s, and 3T3-L1 pCMVneo cell clones were treated with differentiating agents. Nuclear proteins were extracted and separated (20 μg/lane) by SDS–15% PAGE and transferred to polyvinylidene difluoride membranes. Western blots were incubated first with antibodies specific for the HMGI(Y) protein and then with horseradish peroxidase-conjugated secondary antibodies; the immunocomplexes were detected by enhanced chemiluminescence. As a control for equal loading, the blotted proteins were stained with Ponceau Red. The same Western blots were incubated with antibodies to the ubiquitous γ-tubulin protein. Sources of proteins were the following: lane 1, PC MPSV cells (positive control); lane 2, 3T3-L1 cells; lanes 3 and 4, 3T3-L1-HMGI(Y)as cells (clones 1 and 2); lane 5, 3T3-L1 pCMVneo clone 1; lanes 6, 7, and 8, 3T3-L1-HMGI(Y)s cells (clones 1, 2, and 3).

FIG. 3

FIG. 3

(A) Inhibition of adipocytic differentiation induced by blockage of HMGI(Y) synthesis. Adipogenic differentiation of normal and pCMVneo- or pCMV-HMGI(Y)as-transfected 3T3-L1 cells is shown. Cell clones were cultured in the presence of standard differentiation induction medium containing 0.5 mM 1-methyl-3-isobutylxanthine, 1 mM dexamethasone, 5 μg of insulin/ml, and 10% fetal bovine serum. After 8 days of differentiation, cells were observed by light microscopy. Magnification, ×400. This experiment is representative of five independent assays. (B) mRNA levels of aP2 and leptin were determined by RT-PCR, gel electrophoresis, and Southern blot hybridization. For details, see Materials and Methods. The cDNAs were coamplified with GAPDH, as an internal control. No bands are seen in non-reverse-transcribed RNAs, thus excluding DNA contamination (data not shown). RNAs were extracted from these cells at days 0, 4, and 7 of differentiation, as indicated.

FIG. 4

FIG. 4

(A) Cells were grown as described in Materials and Methods and counted daily. (B) Cells were grown until confluence and then induced to differentiate as described in Materials and Methods. Cell counts started 1 day after the addition of the differentiation cocktail, as indicated, and were performed daily. For each type of experiment, one representative clone is shown; the results were confirmed on two additional clones. The data reported are the average results of two independent experiments.

FIG. 5

FIG. 5

(A) Interaction between C/EBPβ and HMGI(Y). Cell lysates were prepared from 3T3-L1 cells at the indicated times as described in Materials and Methods. Proteins were immunoprecipitated with antibodies directed against the protein C/EBPβ (Santa Cruz Biotechnology), as indicated. Immunoprecipitated (I.P.) proteins were immunoblotted with anti-HMGI(Y). Levels of C/EBPβ during differentiation are shown. IgG, immunoglobulin G. (B) Wild-type C/EBPβ protein was in vitro translated as described in Materials and Methods. Rabbit reticulocyte extracts were mixed with GST-HMG-Y recombinant protein for binding assays. Binding reaction products were washed, and proteins were separated on a polyacrylamide gel. Filters were probed with the anti-C/EBPβ antibody (Santa Cruz Biotechnology). (C) C/EBPβ and HMGI(Y) interaction in 293 cells. 293 cells were transfected with the indicated expression plasmids as described in Materials and Methods. Cell lysates were prepared, and equal amounts of proteins were immunoprecipitated with the indicated antibodies. In the top two panels, lane 1 shows a control immunoprecipitation. In lanes 2 and 3, the indicated cell lysates were immunoprecipitated either with the anti-C/EBPβ antibody (upper panel) or with the anti-HMGI(Y) antibody. The immunocomplexes were immunoblotted with the reciprocal antibodies, as indicated. In the bottom two panels, Western blot analysis shows the amounts of C/EBPβ and HMGI(Y) for each lysate used in the top two panels.

FIG. 6

FIG. 6

The region between the second and the third AT hook is required for HMGI(Y)-C/EBPβ interaction. (A) Schematic diagram of plasmids expressing HA-tagged wild-type HMGI(Y), showing the 1- 63, 1-53, and 1-43 deletion mutant proteins. The HA epitope tag, AT hooks (+), and C-terminal (----) domains are also indicated. (B) 293 cells were transiently cotransfected with C/EBPβ and the indicated HMGI(Y) mutant plasmids. Equal amounts of cell lysates (2 mg) were immunoprecipitated with anti-C/EBPβ antibodies, and the immunocomplexes were probed with either anti-C/EBPβ (upper panel) or anti-HA antibodies (lower panel). Aliquots of the same lysates (50 μg) were probed with anti-HA antibodies to evaluate the comparable expression of the transfected plasmids.

FIG. 7

FIG. 7

(A) Leptin transactivation by C/EBPβ and cooperation with HMGI(Y). Histograms show the luciferase activities of extracts from 293 cells cotransfected with the p(−161)ob-luc reporter and the indicated C/EBPβ and HMGI(Y) plasmids. The mutant m52 reporter plasmid was used as a negative control. (B) Histograms showing the luciferase activities of extracts from 293 cells cotransfected with the RSV-luc reporter and the C/EBPβ and HMGI(Y) plasmids. The relative activities were calculated by dividing the normalized activities by the activity of the m52 and RSV-luc constructs, which has been considered equal to 1. The data represent the average of results of three independent experiments, performed in duplicate, with standard deviations. (C) After transfection, cell lysates were divided into two aliquots. One of these aliquots was used for transactivation assays, and the other was used for Western blot analysis as a control of protein expression. Protein extracts were separated by SDS-PAGE, transferred to Immobilon-P, and immunoblotted with the indicated antibodies.

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