TRPC absence induces pro-inflammatory macrophage polarization to promote obesity and exacerbate colorectal cancer

Abstract

During the past half-century, although numerous interventions for obesity have arisen, the condition's prevalence has relentlessly escalated annually. Obesity represents a substantial public health challenge, especially due to its robust correlation with co-morbidities, such as colorectal cancer (CRC), which often thrives in an inflammatory tumor milieu. Of note, individuals with obesity commonly present with calcium and vitamin D insufficiencies. Transient receptor potential canonical (TRPC) channels, a subclass within the broader TRP family, function as critical calcium transporters in calcium-mediated signaling pathways. However, the exact role of TRPC channels in both obesity and CRC pathogenesis remains poorly understood. This study set out to elucidate the part played by TRPC channels in obesity and CRC development using a mouse model lacking all seven TRPC proteins (TRPC HeptaKO mice). Relative to wild-type counterparts, TRPC HeptaKO mice manifested severe obesity, evidenced by significantly heightened body weights, augmented weights of epididymal white adipose tissue (eWAT) and inguinal white adipose tissue (iWAT), increased hepatic lipid deposition, and raised serum levels of total cholesterol (T-CHO) and low-density lipoprotein cholesterol (LDL-C). Moreover, TRPC deficiency was accompanied by an decrease in thermogenic molecules like PGC1-α and UCP1, alongside a upsurge in inflammatory factors within adipose tissue. Mechanistically, it was revealed that pro-inflammatory factors originating from inflammatory macrophages in adipose tissue triggered lipid accumulation and exacerbated obesity-related phenotypes. Intriguingly, considering the well-established connection between obesity and disrupted gut microbiota balance, substantial changes in the gut microbiota composition were detected in TRPC HeptaKO mice, contributing to CRC development. This study provides valuable insights into the role and underlying mechanisms of TRPC deficiency in obesity and its related complication, CRC. Our findings offer a theoretical foundation for the prevention of adverse effects associated with TRPC inhibitors, potentially leading to new therapeutic strategies for obesity and CRC prevention.

Keywords: TRPC HeptaKO; colorectal cancer; gut microbiota; macrophages; obesity.

FIGURE 1

Ablation all seven TRPC proteins induces obesity in mice. (A) Representative pictures of 8-week-old WT and TRPC HeptaKO mice. (B) Body weight of WT and TRPC HeptaKO mice (n = 7–8). (C) Lee’s index of WT and TRPC HeptaKO mice (n = 4–7). (D) Anatomical pictures of eWAT of WT and Hepta-KO mice (Left). Weight of eWAT in WT and TRPC HeptaKO mice (n = 4–7) (Right). (E) Anatomical pictures of iWAT of WT and HeptaKO mice (Left). Weight of iWAT in WT and TRPC HeptaKO mice (n = 4–7) (Right). (F) Representative images and statistical graphs of H&E staining in the fixed eWAT (n = 4–5, three fields/slice). Scale bar: 50 μm. (G) Representative pictures and statistical graphs in Oil red-stained liver sections (n = 4, three fields/slice). The scale bar, 50 μm. In Figure 1B, Two-way ANOVA test was used, whereas for the other figures, Two-tailed Student’s t-test was employed. *p < 0.05, ***p < 0.001.

FIGURE 2

TRPC absence has no significant effects on colon tissues. (A) Representative colon images of WT and TRPC HeptaKO mice (Left). Colon length and colon weight (Right) (n = 6). (B, C) Representative microscopic images in H&E staining and pathological scores in the colon tissues. Scale bar: 50 μm. (n = 5–6). Two-tailed Student’s t-test was used to determine statistical significance between two groups. ns, no significant.

FIGURE 3

TRPC absence inhibits adipose tissue thermogenesis. (A) The serum T-CHO, TG, LDL-C, HDL-C levels in WT and TRPC HeptaKO mice (n = 5–6). (B) Average food intake per three mice for 24 h (n = 4). (C) Fatty acid regulation genes in adipose tissues of mice (n = 8). (D) Representative immunohistochemistry images showing PGC-1α and UCP1 in eWAT (n = 4–6, three fields/slice), Scale bar = 50 μm. Two-tailed Student’s t-test was used to determine statistical significance between two groups. *p < 0.05, **p < 0.01, ***p < 0.001, ns, no significant.

FIGURE 4

TRPC deficiency increases macrophages and inflammatory factors in adipose tissue. (A, B) RT‒PCR results of inflammatory factors and chemokines in adipose tissues of mice (n = 6–8). (C) Live CD45⁺ CD11b⁺ F4/80⁺ macrophages in eWAT of mice were identified by flow cytometry. Left: the representative images; right: the statistical graph of macrophages (n = 3–4). (D) Live CD45⁺ CD11b⁺ F4/80⁺ CD11c⁺ macrophages (M1 macrophages) in iWAT of mice were identified by flow cytometry. Left: the representative images; right: the statistical graph of macrophages (n = 3–4). Two-tailed Student’s t-test was used to determine statistical significance between two groups. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5

Macrophages from TRPC HeptaKO mice exhibit M1 macrophage polarization and inhibit adipose tissue thermogenesis. (A) RT-PCR results of Il6, Nos2, Il12α, and Tnfα in BMDMs under LPS/IFN-γ stimulation (n = 4–6). (B) The expression levels of IL-1β, IL-6, IL-12p70, and TNF-α from supernatant of BMDMs under LPS/IFN-γ or IL-4 stimulation which were measured by ELISA (n = 3). (C) C3H cells were treated with the suspension of M1-BMDM for 24 h. Left: Schematic illustration. Right: The relative expression levels of fatty acid regulation genes in C3H cells (n = 5–6). In Figure 5B, One-way ANOVA was used, whereas for the other figures, Two-tailed Student’s t-test was employed. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 6

Lacking all seven TRPC proteins results in gut microbiota dysbiosis. (A) Representative PAS-stained microscopic images (Left) and quantification of PAS⁺ goblet cells in colonic tissues of WT and TRPC HeptaKO mice (Right). Scale bar: 50 μm. (n = 5–6). (B) PCA analysis (beta diversity) of gut microbiota in WT and TRPC HeptaKO mice (n = 5). (C, D) The composition and relative abundance of gut microbiota at phylum level and genus level in WT and TRPC HeptaKO mice (n = 5). (E, F) Differentially bacteria between TRPC HeptaKO and WT group at phylum and genus (n = 5). (G) Species richness and diversity of gut microbiota in WT and TRPC HeptaKO mice (n = 5). (H) The body weight of WT and TRPC HeptaKO mice that were treated with Abx or water (n = 4–6). In Figure 6H, Two-way ANOVA test was used, whereas for the other figures, Two-tailed Student’s t-test was employed. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 7

Lacking all seven TRPC proteins facilitates the development of AOM/DSS-induced CRC. (A) Schematic illustration of AOM/DSS-induced CRC strategy. (B, C) Body weight and survival rate of AOM/DSS-induced WT and TRPC HeptaKO mice (n = 11–19). (D) Colon length (n = 10–14). (E) Representative images of the colon in each group. (F, G) Tumor number and tumor loads (tumor loads = the sum of diameters of all tumors in colon, n = 10–14). (H) Tumor number (n = 10–14). (I–K) Representative H&E stained and PCNA-images and pathological scores in the colon tissues (n = 5–7, PCNA pathological scores: three fields/slice). (L) Tnfa, Ifng, Il1b, and Nos2 mRNA levels in colorectal tissues of AOM/DSS-induced WT and TRPC HeptaKO mice (n = 8). Two-tailed Student’s t-test was used to determine statistical significance between two groups. *p < 0.05, **p < 0.01, ***p < 0.001.