TRPC1 links calcium signaling to cellular senescence in the protection against posttraumatic osteoarthritis

Abstract

Transient receptor potential channel 1 (TRPC1) is a widely expressed mechanosensitive ion channel located within the endoplasmic reticulum membrane, crucial for refilling depleted internal calcium stores during activation of calcium-dependent signaling pathways. Here, we have demonstrated that TRPC1 activity is protective within cartilage homeostasis in the prevention of cellular senescence-associated cartilage breakdown during mechanical and inflammatory challenge. We revealed that TRPC1 loss is associated with early stages of osteoarthritis (OA) and plays a nonredundant role in calcium signaling in chondrocytes. Trpc1-/- mice subjected to destabilization of the medial meniscus-induced OA developed a more severe OA phenotype than WT controls. During early OA development, Trpc1-/- mice displayed an increased chondrocyte survival rate; however, remaining cells displayed features of senescence including p16INK4a expression and decreased Sox9. RNA-Seq identified differentially expressed genes related to cell number, apoptosis, and extracellular matrix organization. Trpc1-/- chondrocytes exhibited accelerated dedifferentiation, while demonstrating an increased susceptibility to cellular senescence. Targeting the mechanism of TRPC1 activation may be a promising therapeutic strategy in OA prevention.

Keywords: Bone biology; Calcium channels; Cell biology; Cellular senescence; Osteoarthritis.

Figure 1. Analysis of TRPC1 protein expression in human and murine articular cartilage.

(A) Fluorescence images depicting intracellular Ca²⁺ (green) within early passage WT and TRPC1^–/– chondrocytes loaded with Fluo-4 Ca²⁺ indicator, during stimulation with 5 ng/mL thapsigargin. Scale bar: 200 μm. (B) Comparison of basal intracellular Ca²⁺ levels as measured by fluorescence intensity of early passage WT and Trpc1^–/– chondrocytes. Unpaired t test (n = 8). (C–E) Time course analysis of intracellular Ca²⁺ levels as measured by fluorescence intensity of loaded Fluo-4 Ca²⁺ indicator in WT and Trpc1^–/– chondrocytes during stimulation with either ionomycin (200 nM), thapsigargin (5 ng/mL), or bFGF (50 ng/mL). Two-way ANOVA with multiple comparisons (n = 4). *P < 0.05 and **P < 0.01. (F) Immunofluorescence detection of TRPC1 (green) in WT murine chondrocytes in resting conditions and 3 minutes following bFGF (50ng/mL) stimulation. Cell cytoskeleton is counterstained with phalloidin (white) and nuclei with DAPI (blue). Scale bar: 20 μm. For isotype negative control staining, see Supplemental Figure 3A. (G) Immunohistological detection of TRPC1 in human cartilage sections comparing healthy (OARSI score 0), early OA (OARSI score 1.0–2.5) and advanced OA (OARSI score 3.0+). Scale bar: 100 μm. For isotype negative control staining, see Supplemental Figure 3B. (H) Graph showing quantification of TRPC⁺ cells within the cartilage expressed as percentage of total number of cells identified (n = 4 participants). One-way ANOVA with Tukey’s multiple-comparison test. (I) Immunohistological detection of TRPC1 in murine knee joint 2 weeks and 8 weeks following sham control or DMM surgery. Dotted red outline indicates area used for analysis of articular cartilage. Scale bar: 100 μm. For isotype negative control staining, see Supplemental Figure 3C. (J) Quantification of the relative number of TRPC1⁺ cells within the tibial articular cartilage expressed as a percentage of cells present P values: unpaired t test (n = 5 mice).

Figure 2. Assessment of articular cartilage phenotype 8 weeks after surgery (sham versus DMM).

(A) Representative images of Safranin-O–stained paraffin sections of medial compartments of knee joints of WT control and Trpc1^–/– mice 8 weeks after DMM. Scale bar: 100 μm. (B) Summed OARSI score of medial and lateral compartments of WT and Trpc1^–/– knee joints 8 weeks after DMM (n = 7). (C) Immunofluorescence detection of collagen type II in the medial compartments of WT and Trpc1^–/– knee joints 8 weeks after DMM. Scale bar: 100 μm. For isotype negative control staining, see Supplemental Figure 3D. (D) Quantification of the mean fluorescent signal intensity of collagen type II immunofluorescence within the remaining medial articular cartilage (n = 5). For all graphs, data are expressed as mean ± SEM and P values were determined by unpaired t test.

Figure 3. Assessment of early cartilage and subchondral bone changes 2 weeks after DMM.

(A) Representative images of Safranin-O–stained paraffin sections of medial compartments of knee joints of WT control and Trpc1^–/– mice 2 weeks after surgery (sham versus DMM). Scale bar: 100 μm. (B) Summed OARSI score of medial and lateral compartments of WT and Trpc1^–/– knee joints 2 weeks after DMM (n = 8). (C) Bone volume fraction (normalized for total volume; BV/TV) fraction measurement of WT and Trpc1^–/– knee joint tibial subchondral bone 2 weeks after sham control or DMM (n = 8). (D) Trabecular thickness (Tb.th. [mm]) measurement of WT and Trpc1^–/– knee joint tibial subchondral bone 2 weeks after sham control or DMM surgery (n = 8). (E) Trabecular separation (Tb.Sp. [mm]) in WT and Trpc1^–/– knee joint tibial subchondral bone 2 weeks after sham control or DMM (n = 8). (F) Immunofluorescence detection of collagen type II (green) in the medial compartments of WT and Trpc1^–/– knee joints 2 weeks after DMM. Scale bar: 100 μm. For isotype negative control staining, see Supplemental Figure 3D. (G) Quantification of the mean fluorescent signal intensity of collagen type II immunofluorescence within the medial articular cartilage (n = 5). One-way ANOVA with Tukey’s multiple-comparison test. (H) Immunofluorescence detection of collagen type X (red) in the medial compartments of WT and Trpc1^–/– knee joints 2 weeks after DMM. Scale bar: 100 μm. For isotype negative control staining, see Supplemental Figure 3E. (I) Quantification of the mean fluorescent signal intensity of collagen type X immunofluorescence within the medial articular cartilage (n = 5).One-way ANOVA with Tukey’s multiple-comparisons test. For all graphs, data are expressed as mean ± SEM. P values were determined by unpaired t test unless stated otherwise.

Figure 4. TRPC1-dependent phenotypic changes in cartilage during early stages of OA development in mice.

(A) Immunofluorescence detection of Sox9 in the medial compartments of WT and Trpc1^–/– knee joints 2 weeks after DMM. DAPI used as a nuclear counterstain. (B) Quantification of density of Sox9⁺ cells within the medial tibial articular cartilage. One-way ANOVA with Tukey’s multiple-comparison test. (n = 5). (C) Comparison of overall cellularity of medial tibial articular cartilage in WT and Trpc1^–/– 2 week after DMM and sham mice as defined by number of DAPI⁺ cells per 100 μm². One-way ANOVA with Tukey’s multiple-comparison test. (n = 5). (D) Immunofluorescence detection of Ki67 as a marker of cell proliferation in medial compartments of murine knee joints 2 weeks after DMM. DAPI used as a nuclear counterstain. (E) Quantification of number of Ki67⁺ cells within the medial tibial articular cartilage normalized for the total number of DAPI⁺ cells. Unpaired t test (n = 4). (F) Absolute quantification of the number of Ki67⁺ cells and number of DAPI⁺ cells within the medial tibial articular cartilage 2 weeks after DMM. One-way ANOVA with Tukey’s multiple-comparison test (n = 4). (G) TUNEL staining of medial compartments of murine knee joints 2 weeks after DMM. DAPI used as a nuclear counterstain. (H) Quantification of number of TUNEL⁺ cells normalized for total number of cells within the tibial articular cartilage. One-way ANOVA with Tukey’s multiple-comparison test (n = 5). (I) Absolute numbers of TUNEL⁺ and DAPI⁺ cells within the tibial articular cartilage 2 weeks after DMM. (J) Quantification of number of empty lacunae within the tibial articular cartilage 2 weeks after DMM. (K) Immunofluorescence detection of MMP13 in medial compartments of WT and Trpc1^–/– knee joints 2 weeks after DMM. DAPI used as a nuclei counterstain. (L) Quantification of mean MMP13 staining intensity. Unpaired t test (n = 5) unless stated otherwise. Scale bars: 100 μm. For isotype negative control stainings, see Supplemental Figure 3.

Figure 5. RNA expression profiles show an altered response to DMM in cartilage and subchondral bone.

(A) Diagram of RNA-Seq workflow. (B) Volcano plot of cartilage gene expression fold-changes (FC, log₂ scale) in Trpc1^–/– relative to WT mice 2 weeks after DMM and corresponding FDR-adjusted P values (P_adj, –log₁₀ scale). Dashed lines delineate cut-off values (Benjamini-Hochberg FDR, 0.05; FC, 1.5), and red dots highlight significantly differentially expressed genes (DEGs) (i.e., FC > 1.5 with P_adj < 0.05). (C) Heatmap showing DEG profiles (genes with FC > 1.5) in the individual samples from both Trpc1^–/– and WT groups, where green represents upregulation and red represents downregulation. (D) Biological pathway (BP) enrichment dot plot where gene ratio on the x axis represents the ratio of the total DEGs in the given GO term on the y axis. The size of each dot symbolizes the number of DEGs annotated with a specific term.

Figure 6. In vitro analysis of the effect of Trpc1 deficiency upon chondrocyte phenotypic stability.

(A–D) qPCR analysis of sox9, aggrecan, col2a1, and col10a1 gene expression in murine WT and Trpc1^–/– chondrocytes during serial in vitro passage. Two-way ANOVA with Tukey’s multiple-comparison test (n = 3). (E) Representative images of SA–β-Gal staining (blue) of WT and Trpc1^–/– chondrocytes at equivalent confluence, visible by DIC counterimage, during serial passage. Scale bar: 50 μm. (F) Quantification of number of SA–β-Gal⁺ cells normalized for total number of cells during WT and Trpc1^–/– chondrocytes during serial passage. Two-way ANOVA with multiple comparisons (n = 4, 20 images per condition). (G) qPCR analysis of p16INK4a gene expression in WT and Trpc1^–/– chondrocytes during serial passage. Two-way ANOVA with multiple comparisons (n = 3). (H) Western blot of p16^INK4a protein levels in WT and Trpc1^–/– chondrocytes during serial passage. GAPDH used as a loading control. (I) Quantification of Western blotting for p16^INK4a in WT and Trpc1^–/– chondrocytes during serial passage. Two-way ANOVA with multiple comparisons (n = 3).

Figure 7. TRPC1 is required for protection against cellular senescence driven by IL-1β and during murine OA development.

(A) Representative images of SA–β-Gal stainings (blue) of P0 WT and Trpc1^–/– chondrocytes treated with 10 ng/mL IL-1β for either 24 hours, 48 hours, or for 24 hours followed by an additional 24 or 48 hours culture in control medium (rest). (B) Quantification of number of SA–β-Gal⁺ cells normalized to those in control untreated samples (n = 5). (C) CyQUANT fluorescence quantification of WT and Trpc1^–/– chondrocytes measured at 24 and 48 hours after IL-1β stimulation to give a ratio representing proliferation rate (n = 4). (D) Western blot of p16^INK4a protein levels in WT and Trpc1^–/– chondrocytes treated with 10 ng/mL IL-1β for either 24 hours or for 24 hours followed by 48 hours culture in control medium. (E) Quantification of Western blotting for p16^INK4a in WT and Trpc1^–/– chondrocytes treated with 10 ng/mL IL-1β for either 24 hours or for 24 hours followed by 48 hours culture in control medium (n = 3). (F) CCL-2 concentration of control and IL-1β–treated chondrocyte supernatants measured by ELISA (n = 4). (G) Representative images of cleaved caspase activity in chondrocytes treated with IL-1β for 24 hours plus 48 hours rest. (H) Quantification of cleaved caspase activity in WT and Trpc1^–/– chondrocytes following IL-1β stimulation (WT, n = 4; Trpc1^–/–, n = 5). (I) Immunofluorescence detection of p16^INK4a in medial compartments of WT and Trpc1^–/– murine knee joints 2 weeks after DMM. DAPI used as a nuclear counterstain. Scale bar: 100 μm. For isotype negative control staining, see Supplemental Figure 3I. (J) Quantification of number of p16^INK4a+ cells within the medial articular cartilage normalized for the total number of DAPI⁺ cells (n = 5). Two-way ANOVA with multiple comparisons.