Serinc5 Regulates Sequential Chondrocyte Differentiation by Inhibiting Sox9 Function in Pre-Hypertrophic Chondrocytes

Abstract

The growth plate is the primary site of longitudinal bone growth with chondrocytes playing a pivotal role in endochondral bone development. Chondrocytes undergo a series of differentiation steps, resulting in the formation of a unique hierarchical columnar structure comprising round, proliferating, pre-hypertrophic, and hypertrophic chondrocytes. Pre-hypertrophic chondrocytes, which exist in the transitional stage between proliferating and hypertrophic stages, are a critical cell population in the growth plate. However, the molecular basis of pre-hypertrophic chondrocytes remains largely undefined. Here, we employed scRNA-seq analysis on fluorescently labeled growth plate chondrocytes for their molecular characterization. Serine incorporator 5 (Serinc5) was identified as a marker gene for pre-hypertrophic chondrocytes. Histological analysis revealed that Serinc5 is specifically expressed in pre-hypertrophic chondrocytes, overlapping with Indian hedgehog (Ihh). Serinc5 represses cell proliferation and Col2a1 and Acan expression by inhibiting the transcriptional activity of Sox9 in primary chondrocytes. Chromatin profiling using ChIP-seq and ATAC-seq revealed an active enhancer of Serinc5 located in intron 1, with its chromatin status progressively activated during chondrocyte differentiation. Collectively, our findings suggest that Serinc5 regulates sequential chondrocyte differentiation from proliferation to hypertrophy by inhibiting Sox9 function in pre-hypertrophic chondrocytes, providing novel insights into the mechanisms underlying chondrocyte differentiation in growth plates.

Keywords: Serinc5; Sox9; growth plate chondrocyte; pre‐hypertrophic chondrocyte; single‐cell RNA‐seq.

Figure 1

Generation and immunohistochemical analysis of E15.0 Col11a2‐ZsGreen mice. (a) Schematic model to generate Col11a2‐ZsGreen mice. E15.0 embryo photographed using fluorescence microscopy. Scale bar: 1 mm. (b) Immunostaining for Col2 and Col1 in femur from E15.0 Col11a2‐ZsGreen mice. Scale bar: 250 μm. (c) Immunostaining for Col1 in perichondrium from E15.0 Col11a2‐ZsGreen mice femur. Arrowheads indicate Col1‐positive osteoblasts overlapping with ZsGreen. Scale bar: 100 μm. (d) Immunostaining for Col2 and Col1 in interzone from E15.0 Col11a2‐ZsGreen mice femur. Higher magnification images of the boxed area (upper panel) are shown in the lower panel. Scale bar: 100 μm. F: Femur T: Tibia. (e) Immunostaining for Col10 and Mmp13 in hypertrophic chondrocytes from E15.0 Col11a2‐ZsGreen mice femur. Scale bar: 100 μm. HC, hypertrophic chondrocytes; PC, proliferating chondrocytes; RC, round chondrocytes.

Figure 2

Single‐cell RNA‐seq analysis of Col11a2‐enh‐Cre marked mesenchymal cells. (a) Strategy for single‐cell RNA‐seq analysis of growth plate chondrocytes isolated from the limbs of E15.0 Col11a2‐ZsGreen mice. (b) UMAP plots of ZsGreen‐positive cells isolated from the limbs of E15.0 Col11a2‐ZsGreen mice. CH, chondrocyte. (c) Percentage of total cell numbers in each cluster. (d) Ridgeline plot showing Sox9, Col2a1, and Col1a1 expression. (e) Violin plot showing marker gene expression in chondrocytes and osteoblasts. (f) Expression patterns of chondrocyte and osteoblast marker genes in UMAP plots. Arrowheads indicate cluster 5. UMAP, uniform manifold approximation and projection.

Figure 3

Identification of Serinc5 as the specific marker gene for pre‐hypertrophic chondrocytes. (a) List of 10 significant differentially expressed genes (DEGs) in cluster 5. (b) Violin plot showing the top 10 DEGs in cluster 5. (c) Haematoxylin and eosin (HE) staining and in situ hybridization analysis of Ihh, Serinc5, and Col10a1 in growth plate chondrocytes from E15.0 mice tibia. Scale bar: 200 μm (d) Feature plot illustrating the expression of Serinc5 and Ihh. Arrowheads indicate cluster 5. (e) Violin plot showing the expression of Serinc family genes.

Figure 4

Expression pattern of Serinc5 during chondrocyte differentiation. The flowchart upper in the panel illustrates the process of micromass culture of mouse limb bud cells. The graphs indicate gene expression of chondrocyte markers and Serinc5, determined using RT‐qPCR. Data are shown as fold activation normalized to day 0 (means ± SD, n = 3).

Figure 5

Inhibition of chondrocyte proliferation and differentiation by Serinc5. (a) Proliferation of primary chondrocytes infected with Empty Vector (EV) or Serinc5 lentivirus cultured for 2 days in a 96‐well plate, measured using the WST‐1 Proliferation Assay Kit (n = 3, Student's t‐test). (b, c) Total RNA in primary chondrocytes infected with EV or Serinc5 lentivirus cultured with or without recombinant BMP2 analyzed by RT‐qPCR for marker genes of resting/proliferating chondrocytes (b) and pre‐hypertrophic/hypertrophic chondrocytes (c). Data are shown as the mean ± SD. (n = 3, biologically independent samples). Statistical analysis was performed using one‐way ANOVA followed by Tukey's multiple comparison test.

Figure 6

Inhibition of Sox9‐dependent chondrocyte gene expression by Serinc5. (a) Immunoblot analysis illustrating the expression levels of Serinc5, Sox9, and β‐actin in primary chondrocytes infected with Empty Vector (EV) or Serinc5 lentivirus, followed by Sox9 overexpression. (b) RT‐qPCR results showing the relative mRNA levels of Col2a1 and Acan in primary chondrocytes infected with EV or Serinc5 lentivirus followed by Sox9 overexpression. Data are shown as the mean ± SD. (n = 3, biologically independent samples). **p < 0.01, one‐way ANOVA followed by Tukey's multiple comparison test. (c) Luciferase assay results demonstrating the transcriptional activity of Col2a1 and Acan in C3H10T1/2 cells transfected with Col2a1 or Acan luciferase construct along with EV, Serinc5, or Sox9 expression vectors. Data are shown as the mean ± SD. (n = 4, biologically independent samples). **p < 0.01, one‐way ANOVA followed by Tukey's multiple comparison test.

Figure 7

Epigenetic regulation of Serinc5 in chondrocytes. (a) ATAC‐seq and ChIP‐seq profiles of the genomic region around mouse Serinc5 gene in mesenchymal stem cells and primary chondrocytes. Light gray shading magnified in the lower panel highlights the candidate genomic region of the Serinc5 enhancer in chondrocytes. (b) Luciferase reporter constructs of deletions of Serinc5 enhancer were transfected into primary chondrocytes. Luciferase activities were measured at 48 h after transfection. Data are shown as the mean ± SD. (n = 4, biologically independent samples). (c) Total RNA isolated from ATDC5 treated with or without ITS was analyzed by RT‐qPCR for Serinc5 gene expression. Data are shown as the mean ± SD. (n = 3, biologically independent samples). Unpaired Student's t‐test (d) ChIP‐qPCR analysis of Serinc5 enhancer in ATDC5 cells treated with or without ITS. Sonicated chromatin isolated from ATDC5 cells was immunoprecipitated with an anti‐H3K27ac antibody and quantified using specific primers for Serinc5 enhancer. Data are shown as the mean ± SD. (n = 3, biologically independent samples). Unpaired Student's t‐test. ITS, insulin, transferrin, selenium.