Transcription factor 21 (TCF21) promotes proinflammatory interleukin 6 expression and extracellular matrix remodeling in visceral adipose stem cells

Abstract

The visceral (VIS) and subcutaneous (SQ) fat pads are developmentally distinct white adipose tissue depots and contribute differently to inflammation and insulin resistance associated with obesity. The basic helix-loop-helix transcriptional regulator, transcription factor 21 (TCF21), is a marker gene for white adipose tissues and is abundantly expressed in VIS-derived adipose stem cells (ASCs), but not in SQ-derived ASCs. However, TCF21's role in regulating fat depot-specific gene expression and function is incompletely understood. Here, using siRNA-mediated Tcf21 knockdowns and lentiviral gene transfer of TCF21 in mouse ASCs, we demonstrate that TCF21 is required for the VIS ASC-specific expression of interleukin 6 (IL6), a key cytokine that contributes to the proinflammatory nature of VIS depots. Concurrently, TCF21 promotes MMP-dependent collagen degradation and type IV collagen deposition through the regulation of the extracellular matrix (ECM) modifiers, matrix metalloproteinase (MMP) 2, MMP13, and tissue inhibitor of MMP1 (TIMP1), as well as collagen type IV α1 chain (COL4A1) in VIS ASCs. We also found that although IL6 mediates the expression of Mmp13 and Timp1 in VIS ASCs, the TCF21-dependent expression of Mmp2 and Col4a1 is IL6-independent. These results suggest that TCF21 contributes to the proinflammatory environment in VIS fat depots and to active ECM remodeling of these depots by regulating IL6 expression and MMP-dependent ECM remodeling in a spatiotemporally coordinated manner.

Keywords: adipose tissue; collagen; interleukin 6 (IL6); matrix metalloproteinase (MMP); transcription.

Figure 1.

TCF21 is expressed predominantly in visceral WAT ASCs. A and B, vascular stromal cells were isolated from visceral and subcutaneous adipose depots and then subjected to magnetic cell sorting using anti-Sca1 antibody to enrich Sca1-high ASCs. A, a volcano plot of gene expression in VIS and SQ ASCs. B, total RNA samples were prepared from VIS and SQ ASCs and analyzed with RT-qPCR (n = 3 independent biological replicates, means ± S.E.). *, p < 0.05; ***, p < 0.001; ****, p < 0.0001 by Student′s t test. C, RT-qPCR with human primary preadipocytes (n = 3 independent biological replicates, means ± S.E.). *, p < 0.05 by Student's t test. D, immunostaining of endogenous TCF21 (red), nucleus (DAPI, blue), and F-actin (green) in VIS and SQ ASCs. Scale bar, 50 μm. E, immunostaining of endogenous TCF21 (red), IL6 (green), and DAPI (blue) in unfractionated vascular stromal cells. Scale bar, 20 μm. The right panel shows the percentage of each group of cells per total vascular stromal cells, means ± S.E. F, left panel, immunostaining of endogenous TCF21 (red), PDRGFRα (green), and nucleus (DAPI, blue); right panel, TCF21 (red), IL6 (green), and nucleus (DAPI, blue) in VIS and SQ fat depots in vivo. Cells expressing both TCF21 and PDGFRα or IL6 were indicated by arrows. Scale bar, 20 μm.

Figure 2.

TCF21 drives IL6 expression in VIS ASCs. A and B, the siRNA oligonucleotides targeting Tcf21 and control siRNA were transfected to mouse VIS ASCs and cultured for 3 days. Total RNA was extracted to assess Tcf21 and Il6 expression by RT-qPCR (n = 4 independent biological replicates, means ± S.E.). *, p < 0.05; ***, p < 0.001; ****, p < 0.0001 by one-way ANOVA. C, IL6 and type I collagen (COL1) protein levels in the conditioned media of siRNA transfected VIS and SQ ASCs. Conditioned media were collected at indicated time points for Western blotting. Independent experiments were performed twice. D, mouse VIS ASCs were transduced with human TCF21 cDNA by lentiviral gene transfer. Total RNA was extracted for RT-qPCR analysis of TCF21 and Il6 expression (n = 3 independent biological replicates, means ± S.E.). *, p < 0.05 by ratio paired t test. E, schematic diagram of human IL6 promoter region cloned into pGL3-basic reporter plasmid. Known cis-elements (black boxes) and predicted TCF21-binding E-box sites (hatched boxes) are shown along with respective sequences. F, IL6 promoter in CHO cells regulated by co-transfected full-length and mutant TCF21. Reconstituted IL6 promoter activity (firefly luciferase) was normalized by co-transfected thymidine kinase promoter activity (Renilla luciferase; n = 7, means ± S.E.). ****, p < 0.0001 by one-way ANOVA. The experiment was independently repeated.

Figure 3.

TCF21 regulates the expression of a subset of MMPs and TIMPs. The siRNA oligonucleotides targeting Tcf21 and control siRNA were transfected to mouse VIS ASCs and cultured for 3 days. A, total RNA was extracted for RT-qPCR analysis (n = 4, means ± S.E.). **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 by one-way ANOVA. B, to assess MMP2 activity, the media of transfected cells were changed to serum-free DMEM a day after transfection; conditioned media were collected 3 days later and subjected to gelatin zymography. This is a representative figure (n = 3, means ± S.E.). ****, p < 0.0001 by one-way ANOVA.

Figure 4.

TCF21 mediates type IV collagen deposition by VIS ASCs. A–C, mouse VIS ASCs were transfected with siRNA oligonucleotides targeting Tcf21 and a control oligonucleotide and then cultured for 3 days. A, RT-qPCR analysis of collagen gene expression (n = 4, means ± S.E.). *, p < 0.05; **, p < 0.01 by one-way ANOVA). B, extracellular type IV collagen was detected (red) and quantified, normalized by the number of nuclei (blue). Scale bar, 50 μm (low mag) and 10 μm (high mag) (n = 5, means ± S.E., not significant per two-way ANOVA). C, extracellular degraded collagen was stained (red) and quantified per nuclei (blue) (n = 5, means ± S.E.) Scale bar, 50 μm. *, p < 0.05; ****, p < 0.001 by two-way ANOVA. D, ASCs isolated from VIS WAT were cultured in the presence or absence of 10 μm GM6001 for 2 days, stained for degraded collagen (red), and quantified per nuclei (blue) (n = 5, means ± S.E.). Scale bar, 50 μm. *, p < 0.05 by Student's t test. E and F, ASCs isolated from VIS and SQ WATs were transduced with TCF21 cDNA by lentiviral gene transfer. The cells were cultured for 3 days and analyzed by immunostaining for collagens type IV, I, and VI and degraded type I collagen (all in red) along with DAPI (nuclei, blue). Scale bar, 50 μm. Signal intensities of staining were quantified and normalized by nucleus number (n = 5, means ± S.E.). *, p < 0.05; **, p < 0.01; ****, p < 0.001 by two-way ANOVA (E) or by Student's t test (F).

Figure 5.

IL6 is responsible for the expression of Mmp13 and Timp1 but not for type IV collagen deposition and type I collagen degradation. A, VIS and SQ ASCs were incubated in the presence and absence of IL6-neutralizing antibody (IL6 Ab, 100 ng/ml) with and without 30 ng/ml recombinant mouse IL6 for 24 h. Total RNA was isolated 24 h later and analyzed by RT-qPCR (n = 3, means ± S.E.). *, p < 0.05 by two-way ANOVA. B, mouse VIS ASCs were transfected with siRNA oligonucleotides targeting Tcf21 and a control oligonucleotide and then treated with IL6-neutralizing antibody (100 ng/ml) for 3 days. Extracellular deposition of type IV collagen and degraded collagen products are shown in red, and nuclei are in blue. Scale bar, 50 μm. Signal quantification is shown on the right (n = 5, means ± S.E.). **, p < 0.01 by two-way ANOVA.