Tight regulation of wingless-type signaling in the articular cartilage - subchondral bone biomechanical unit: transcriptomics in Frzb-knockout mice

Abstract

Introduction: The aim of this research was to study molecular changes in the articular cartilage and subchondral bone of the tibial plateau from mice deficient in frizzled-related protein (Frzb) compared to wild-type mice by transcriptome analysis.

Methods: Gene-expression analysis of the articular cartilage and subchondral bone of three wild-type and three Frzb-/- mice was performed by microarray. Data from three wild-type and two Frzb-/- samples could be used for pathway analysis of differentially expressed genes and were explored with PANTHER, DAVID and GSEA bioinformatics tools. Activation of the wingless-type (WNT) pathway was analysed using Western blot. The effects of Frzb gain and loss of function on chondrogenesis and cell proliferation was examined using ATDC5 micro-masses and mouse ribcage chondrocytes.

Results: Extracellular matrix-associated integrin and cadherin pathways, as well as WNT pathway genes were up-regulated in Frzb-/- samples. Several WNT receptors, target genes and other antagonists were up-regulated, but no difference in active β-catenin was found. Analysis of ATDC5 cell micro-masses overexpressing FRZB indicated an up-regulation of aggrecan and Col2a1, and down-regulation of molecules related to damage and repair in cartilage, Col3a1 and Col5a1. Silencing of Frzb resulted in down-regulation of aggrecan and Col2a1. Pathways associated with cell cycle were down-regulated in this transcriptome analysis. Ribcage chondrocytes derived from Frzb-/- mice showed decreased proliferation compared to wild-type cells.

Conclusions: Our analysis provides evidence for tight regulation of WNT signalling, shifts in extracellular matrix components and effects on cell proliferation and differentiation in the articular cartilage - subchondral bone unit in Frzb-/- mice. These data further support an important role for FRZB in joint homeostasis and highlight the complex biology of WNT signaling in the joint.

Figure 1

Microarray analysis of cartilage and subchondral bone. (A) Frontal hematoxylin-safranin O stained section of the tibia articular cartilage and subchondral bone isolated from a wild-type C57Bl/6 mouse at six weeks of age. Dissected tissues include the articular cartilage (AC), the underlying subchondral bone (SB) containing trabeculae and bone marrow and the upper part of the growth plate (GP). Scale bar = 50 μm (B) Heatmap showing the correlation between the Robust Multiarray Averaging (RMA) expression values for all samples. The three wild-type (WT) and one heterozygous frizzled-related protein (Frzb^+/-) (HZ) mouse cluster apart from the two Frzb^-/- (KO) mice. Correlations are presented by colors going from green (lowest) to red (highest). (C) Analysis of the representation of genes in the microarray associated with articular cartilage (collagen type 2a1 (Col2a1), collagen type 9a1 (Col9a1), aggrecan, chondromodulin, cartilage oligomeric matrix protein (Comp)), bone (osteopontin, osteocalcin, collagen type 1a1 (Col1a1), bone sialoprotein 2 (Ibsp), tartrate-resistant acid phosphatase type 5 (Acp5)), and hematopoiesis (B-lymphocyte antigen CD20 (Ms4a1), B-cell progenitor kinase (Btk), lymphoid-restricted immunoglobulin octamer-binding protein (Pou2f2), CD3 antigen (CD3), CD8 antigen (CD8), protein tyrosine phosphatase, receptor type C (CD45)). Cartilage- and bone-specific genes were found in the highest percentiles, while T cell, B cell and platelet related genes, were found in lower amounts.

Figure 2

Molecular analysis of the articular cartilage - subchondral bone to corroborate the microarray data. (A) Real-Time PCR analysis of tibia articular cartilage and subchondral bone from frizzled-related protein-knockout (Frzb^-/-) mice compared to wild-types. Frzb was virtually absent and secreted frizzled-related protein 1 (Sfrp1) and secreted frizzled-related protein 2 (Sfrp2) were significantly upregulated in Frzb^-/- samples compared to wild-type samples. There was a trend of up-regulation for dickkopf homolog 2 (Dkk2) (one outlier). Data are shown as relative expression values versus hypoxanthine guanine phosphoribosyl transferase (Hprt) (2^-ΔCt) (n = four Frzb^-/- and five wild-type samples; All experiments were performed in duplicate; Mann-Whitney test: P = 0.016 for Frzb, P = 0.032 for Sfrp1, P = 0.016 for Sfrp2 and P = 0.2 for Dkk2). (B-C) Western blot and densitometry analysis of proteins extracted from tibia articular cartilage and subchondral bone showed no consistent change in active (dephospho) β-catenin (80 kDa) (B) and phosphorylated (phospho) Smad 1/5/8 (mothers against decapentaplegic homolog) (55 kDa) (C) between three wild-type (lane 1-3) and in three Frzb^-/- (lane 4-6) samples. Anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (37 kDa) Western blot is shown as loading control. Quantitative analysis was performed with Image J software. Data are shown as the ratio of the mean optical density (OD) for β-catenin or P-Smad/the mean OD of GAPDH (n = three samples/group; Mann-Whitney test: P > 0.05 for β-catenin and for P-Smad).

Figure 3

Chondrogenesis after gain or loss of FRZB in ATDC5 cells. (A) Real-Time PCR analysis showed increased expression of collagen type 2a1 (Col2a1) and aggrecan in the micro-masses overexpressing frizzled-related protein (FRZB) compared to micro-masses expressing control pcDNA3.1+ vector. Data are shown as the mean of the fold difference compared to the control condition at Day 7 normalised to hypoxanthine guanine phosphoribosyl transferase (Hprt) (2^-ΔΔCt) ± SEM (n = six samples/condition; Mann-Whitney test: for aggrecan P = 0.002, P = 0.015 and P = 0.002 and for Col2a1 P = 0.03, P = 0.3 and P = 0.002). (B) Picrosirius Red and Safranin O staining at Day 7 showed increased spreading of collagen fibers and sulphated glycosaminoglycans (GAGs) from the center in micro-masses overexpressing FRZB compared to controls. (C) Protein quantification (optical density (OD) measured at 570 nm) of the micro-masses was comparable between the two groups (n = three samples/group; Mann-Whitney test: P > 0.05). (D) Staining intensity was comparable between FRZB overexpressing micro-masses and controls for Picrosirius Red and significantly decreased for FRZB overexpressing micro-masses for Safranin O staining. Data are shown as the mean OD normalised to the mean protein content (n = three samples/group; Mann-Whitney test: P > 0.05 for Picrosirius Red and P = 0.02 for Safranin O). (E) Real-Time PCR analysis showed decreased expression of Col2a1 and aggrecan in the micro-masses where Frzb was knocked down using the pGIPZ-shRNAmir directed against Frzb compared to controls. Data are shown as the mean of the fold difference compared to the control condition at Day 7 normalised to Hprt (2^-ΔΔCt) ± SEM (n = two to three samples for pGIPZ-Ctrl and five to six samples for pGIPZ-FRZB; Mann-Whitney test: for aggrecan P = 0.02 and for Col2a1 P = 0.02). At Day 14 and Day 21 for pGIPZ-Ctrl n = 2 precluding statistical analysis.

Figure 4

Minor collagen expression after gain or loss of FRZB in ATDC5 cells. (A) Real-Time PCR analysis for collagen type 3α1 (Col3a1) and collagen type 5α1 (Col5a1) expression in the ATDC5 micro-masses overexpressing frizzled-related protein (FRZB) compared to controls at Day 7. Data are shown as the mean fold difference compared to the control condition normalised to hypoxanthine guanine phosphoribosyl transferase (Hprt) (2^-ΔΔCt) ± SEM (n = six samples/condition; Mann-Whitney test: for Col3a1 P = 0.24 and for Col5a1 P = 0.06). (B) RT-PCR analysis for Col3a1 and Col5a1 expression in the ATDC5 micro-masses where Frzb was knocked down using the pGIPZ-shRNAmir directed against Frzb compared to controls at Day 7. Data are shown as the mean fold difference compared to the control condition normalised to Hprt (2^-ΔΔCt) ± SEM (n = six samples for pGIPZ-Ctrl and three samples for pGIPZ-FRZB; Mann-Whitney test: for Col3a1 P = 0.047 and for Col5a1 P = 0.54). (C) Proliferation assay of ribcage articular chondrocytes isolated from Frzb^-/- compared to wild-type (WT) mice. Data are shown as the difference in fluorescence after 24 h and one week. (n = nine conditions/group; Initial cell densities were 500 (black dots), 2,000 (grey dots) and 4,000 (black circles) cells per well; Mann-Whitney test: P = 0.0019, P = 0.0012 and P = 0.0008).