Figure 1 Increased CAV1 mRNA and protein levels in AT. (A). CAV1 mRNA expression in AT isolated from lean and obese individuals, as determined by RT-qPCR assay (B). Representative images of IHC staining for CAV1 protein in tissue sections of AT obtained from lean and obese individuals. Obese sections show stronger staining than lean sections (images, magnification, ×20). (C). IOD/area quantification of CAV1 expression in AT from lean and obese individuals. CAV1 protein expression in obese individuals is two-fold higher, as compared to lean participants. Abbreviations: AT, adipose tissue; CAV1, caveolin-1; IHC, immunohistochemistry; IOD, integrated optical density; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Figure 2 Gene expression and immunohistochemical results in AT isolated from lean and obese individuals. Correlation between CAV1 and (A). TNF-α and (B). NF-κB transcripts expression. (C). Representative images of IHC staining for TNF-α protein in tissue sections of AT obtained from lean and obese individuals. Obese sections show stronger staining than lean sections (images, magnification, ×20. (D). IOD/area quantification of TNF-α expression in AT from lean and obese individuals. TNF-α protein expression in obese individuals is three-fold higher compared to lean participants. (E). CAV1 and TNF-α immunohistochemistry IOD/area correlation. Immunohistochemical analysis was performed on samples from five individuals, and each point is the average of five different images. Abbreviations: AT: adipose tissue; CAV1: caveolin-1; TNF-α: tumor necrosis factor-alpha; NF-κB: nuclear factor kappa-light-chain-enhancer of activated B cells; IHC, immunohistochemistry, IOD: integrated optical density.

Figure 3 Exogenous administration of TNF-α induces CAV1 expression in vivo. (A). The levels of mRNA of CAV1 in subcutaneous adipose tissue obtained from wild-type and TNF-α knock-out mice injected intraperitoneally with saline or TNF-α solution (100 µg/kg). Data are presented as mean ± SEM values of four animals/group. Two-tailed unpaired Student’s t-test was used to determine significance. (B,C). Western blots for total protein extracts (10 µg) were developed with antibodies against CAV1 and β-actin, as indicated. CAV1 expression was significantly elevated in response to TNF-α treatments. Two-tailed unpaired Student’s t-test was used to determine significance. Statistical analyses were relative to untreated wild-type animals (n = 4).

Figure 4 TNF-α augments the expression of CAV1 in differentiated adipocytes isolated from lean and obese individuals. (A). Relative to untreated lean adipocytes, CAV1 mRNA expression levels were significantly elevated in obese adipocytes, and in response to TNF-α treatment for 24 h. Data are presented as mean ± SEM values obtained from three independent experiments (n = 3); a Two-tailed unpaired Student’s t-test was used to determine significance. (B,C). Western blots for total protein extracts (5 µg) were developed with antibodies against CAV1 and β-actin, as indicated. CAV1 basal protein expression was significantly elevated in obese compared to lean adipocytes and was significantly induced in response to TNF-α treatments of both lean and obese adipocytes. A two-tailed unpaired Student’s t-test was used to determine significance (n = 3). * p < 0.001; ** p < 0.05. (D). Treatment with TNF-α activates the NF-κB signaling pathway in both lean and obese adipocytes. Western blots were developed with antibodies against phospho-NF-κB, total NF-κB, IκBα, and the TNF-α receptor TNFR1. 24 h post-TNF-α treatment, NF-κB was activated by phosphorylation and the degradation of its inhibiter IκBα. Furthermore, TNFR1 was desensitized to TNF-α signaling post-24 h treatments (representative Western blots, n = 2).

Figure 5 Treatment with TNF-α is associated with an enhanced CAV1 promoter activation. (A). CAV1 transcript levels were significantly elevated in obese pre-adipocytes relative to the untreated lean pre-adipocytes and in response to TNF-α treatment for 24 h. Data from three independent experiments (n = 3) are presented as mean ± SEM; significance was determined using a two-tailed unpaired Student’s t-test. (B). Western blots for total protein extracts (10 µg) were developed with antibodies against CAV1 and β-actin, as indicated. CAV1 basal protein expression was notably elevated in response to TNF-α treatments in lean pre-adipocytes and in obese compared to lean pre-adipocytes. (C). Schematic representation of the human CAV1 locus showing the regulatory region (vertical box) containing NF-κB binding consensus located 1000 bp upstream of the translation start site (arrow). (D,E). ChIP was performed in pre-adipocytes from lean individuals, either treated with TNF-α or not, and obese individuals, using antibodies against NF-κB or H3K14ac and analyzed by qPCR with primers flanking the NF-κB binding site, as indicated. White bars indicate precipitation with IgG-nonspecific antibodies. Relative enrichment was calculated as the percent chromatin input normalized to IgG from three biological replicas (n = 3). ** p < 0.01. (F). A schematic representation of the CAV1 gene mapping the proximal regulatory region and showing the cloned 250 bp DNA fragment spanning the NF-κB binding sites into the Luciferase-pRMT-Luc reporter vector. (G). Luciferase reporter assays: pre-adipocytes isolated from lean and obese individuals were transfected with a CAV1-5′UTR-Luc construct and were either untreated (black bars) or treated with TNF-α. Luciferase reporter activity was notably higher in samples treated with TNF-α. Luciferase activities are presented as a fold change relative to the pRMT-Luc reporter. Data are presented as mean ± SD of three biological replicas (total n = 9); a two-tailed unpaired Student’s t-test was used to determine significance. * p < 0.05.

Figure 6 In adipocytes, CAV1 gene expression is regulated by TNF-α/TNFR signaling pathways though the activation of the downstream NF-κB pathway, which in turn binds to its consensus site at the CAV1 regulatory promoter and enhances histone acetylation. Created with BioRender.com.