Mohawk is a transcription factor that promotes meniscus cell phenotype and tissue repair and reduces osteoarthritis severity

Abstract

Meniscus tears are common knee injuries and a major osteoarthritis (OA) risk factor. Knowledge gaps that limit the development of therapies for meniscus injury and degeneration concern transcription factors that control the meniscus cell phenotype. Analysis of RNA sequencing data from 37 human tissues in the Genotype-Tissue Expression database and RNA sequencing data from meniscus and articular cartilage showed that transcription factor Mohawk (MKX) is highly enriched in meniscus. In human meniscus cells, MKX regulates the expression of meniscus marker genes, OA-related genes, and other transcription factors, including Scleraxis (SCX), SRY Box 5 (SOX5), and Runt domain-related transcription factor 2 (RUNX2). In mesenchymal stem cells (MSCs), the combination of adenoviral MKX (Ad-MKX) and transforming growth factor-β3 (TGF-β3) induced a meniscus cell phenotype. When Ad-MKX-transduced MSCs were seeded on TGF-β3-conjugated decellularized meniscus scaffold (DMS) and inserted into experimental tears in meniscus explants, they increased glycosaminoglycan content, extracellular matrix interconnectivity, cell infiltration into the DMS, and improved biomechanical properties. Ad-MKX injection into mouse knee joints with experimental OA induced by surgical destabilization of the meniscus suppressed meniscus and cartilage damage, reducing OA severity. Ad-MKX injection into human OA meniscus tissue explants corrected pathogenic gene expression. These results identify MKX as a previously unidentified key transcription factor that regulates the meniscus cell phenotype. The combination of Ad-MKX with TGF-β3 is effective for differentiation of MSCs to a meniscus cell phenotype and useful for meniscus repair. MKX is a promising therapeutic target for meniscus tissue engineering, repair, and prevention of OA.

Fig. 1.. MKX gene expression in meniscus.

(A) MKX expression (arrow) compared to tissues in GTEx and RNA-seq data from meniscus and articular cartilage. Box plots are shown for MKX expression values (logCPM) in 37 tissues in GTEx and in meniscus and cartilage. (B) MKX expression in different joint tissues and BM-MSCs. Human knee joints from healthy donors were collected at autopsy and used for RNA isolation from ACL (n=8), meniscus (n=7), and articular cartilage (n=6), and human BM-MSCs (n=8) from monolayer culture in basal media. qRT-PCR was performed for MKX and GAPDH. P < 0.05 (One-way ANOVA) as compared to meniscus. (C) Immunohistochemistry of MKX in the avascular (AVAS) and vascular (VAS) region of healthy (16/M, Grade 0) and degenerated (82/M, Grade 3) human meniscus tissue. Scale bars, 100 µm. (D) MKX positive cells were quantified and statically analyzed from each avascular and vascular of normal or degenerated human meniscus (n=4 per group). P < 0.05 (One-way ANOVA) compared to the selected groups. (E) RNA-seq analysis of Iroquois (Irx) family-related homeobox protein including MKX (Irx-like 1) from human meniscus (n=10). P < 0.05 (One-way ANOVA) as compared to MKX. All values are mean ± s.e.m.

Fig. 2.. Effects of growth factors and Ad-MKX on gene expression in MSCs.

Human bone marrow derived mesenchymal stem cells (BM-MSCs) (n=4) were cultured with growth factors BMP12, GDF5, and CTGF (100 ng/ml). Ad-MKX-transduced BM-MSCs were cultured with basal media. RNA was isolated after 14 days for qRT-PCR analysis of (A) MKX (B) COL1A1, (C) SCX, (D) TNC, and (E) TNXB. P < 0.05 (One-way ANOVA) as compared to BM-MSC which was normalized to 1. For analysis of protein expression, BM-MSCs (n=4 for each condition) were cultured for 14 days for western blotting analysis of (F) MKX, (G) COL1A1, (H) COL2A1, and (I) ACAN. P < 0.05 (One-way ANOVA) as compared to Ad-GFP transduced BM-MSCs. All values are mean ± s.e.m.

Fig. 3.. Gene expression analysis and immunohistochemistry in BM-MSCs after Ad-MKX transduction and TGF-β3 stimulation.

BM-MSC pellets (n=6) cultured in media with chondrogenic supplements with or without TGF-β3 and with or without Ad-MKX (MOI 100) transduction. RNA was isolated after 2 weeks and analyzed by qRT-PCR. Gene expression of (A) MKX and fibrogenic differentiation related genes; (B) Chondrogenic differentiation related genes; (C) Proteoglycan of meniscus related genes; (D) Meniscus hypertrophy related genes. Immunohistochemistry of COL1A1, COL2A1, and ACAN; (E) BM-MSC pellets in basal media and BM-MSC pellets in TGF-β3 media; (F) Ad-MKX-transduced BM-MSC pellets in basal media and Ad-MKX-transduced BM-MSC pellets in TGF-β3 media. Results are from 3–4 separate experiments, each performed in duplicate. Gene expression was normalized by BM-MSC gene expression. P < 0.05 (One-way ANOVA) as compared to the selected group. All values are mean ± s.e.m. Scale bars: 100 µm.

Fig. 4.. Ad-MKX for overexpression and siRNA mediated knockdown of MKX in meniscus cells.

qRT-PCR analysis of genes related to meniscus degeneration, hypertrophy, and calcification at 48-hour post transduction of meniscus cells (n=6–10) with Ad-MKX or MKX siRNA. (A) Ad-MKX transduction of normal avascular cells. P < 0.05 (One-way ANOVA) as compared to Ad-GFP transduced healthy avascular cells; (B) siRNA MKX transduction of normal avascular cells. P < 0.05 (One-way ANOVA) as compared to siRNA control transduced normal avascular cells; (C) Ad-MKX transduction of OA avascular cells. P < 0.05 (One-way ANOVA) as compared to Ad-GFP-transduced OA avascular cells; (D) Ad-MKX transduction of OA avascular cells with IL-1β (0.5 ng/ml). P < 0.05 (One-way ANOVA) as compared to Ad-GFP-transduced OA avascular cells with IL-1β (0.5 ng/ml) after normalized by Ad-GFP-transduced OA avascular cells without IL-1β. All values are mean ± s.e.m.

Fig. 5.. MKX in the treatment of experimental tears in meniscus explants.

Experimental tears in bovine meniscus explants (n=4) were inserted with various preparations of DMS and cultured for 2 weeks. Groups included DMS only, MSCs on DMS, MSCs seeded on TGF-β3-coated DMS, Ad-MKX-transduced MSCs seeded on DMS, and Ad-MKX-transduced MSCs seeded on TGF-β3-coated DMS. (A) Tissue sections were stained with Safranin-O, DAPI, picrosirius red with polarized light, and differential interference contrast (DIC) imaging. Double headed arrows in Safranin-O and DAPI stain indicate tissue identity. Yellow: bovine meniscus explant; red: conditional DMS. The red fluorescence represents TGF-β3. Arrows in picrosirius red stain and DIC images indicate tissue connectivity conditions. Yellow: non-connected area; orange: connected area with visible borderline; red: connected area and covered with fibers which is shown as white lines; blue: type III collagen fibers which is indicated as a green polarized color covering borderline. (B) The Safranin-O positive area in the DMS was detected by analyzing threshold color and total area and was calculated using Image J. The percent Safranin-O positive area was calculated. (C) DAPI positive cells in the DMS were counted and cell counts were normalized by the DMS and DMS/meniscus interface area that was analyzed. (D) The area of the meniscus/DMS interface that was connected by collagen fibers was measured and expressed as % interconnectivity. Meniscus explants with experimental tears were inserted with various DMS preparations as indicated and cultured for 4 weeks. (E) Tensile testing (n=6) was performed and calculated with Young’s modulus. Data represent a minimum of 3 separate experiments, where each condition was tested in 8 replicates. P < 0.05 (One-way ANOVA) as compared to the selected group. All values are mean ± s.e.m. Scale bars: 100 µm.

Fig. 6.. Ad-MKX injection into mouse knee joints after DMM surgery.

Ad-GFP or Ad-MKX virus (1x10¹⁰ pfu/ml) was injected into the mouse knee joint (n=11; 6 males and 5 females) at 1 week after DMM surgery. The same amount (10 µl) of virus was injected once per week for 4 weeks. (A) Safranin-O staining and immunohistochemistry (COL10A1, and RUNX2) of the mouse knee joint (anterior region) from sham surgery, Ad-GFP-injected knee joint with DMM surgery, and Ad-MKX-injected knee joint with DMM surgery after 4 weeks. “M” indicates degenerative anterior meniscus in male mice. Meniscus histopathological score was calculated for (B) male and (C) female mice, and (D) total scores were compared. Articular cartilage histopathological score was calculated for (E) male and (F) female mice, and (G) total scores were compared. (H) Synovium scores. P < 0.05 (Mann-Whitney test) as compared to the Ad-GFP injected mice group. All values are mean ± s.e.m. Scale bars:100 µm.

Fig. 7.. Ad-MKX injection into human meniscus explants from patients with OA.

Ad-GFP or Ad-MKX virus (1x10¹⁰ pfu/ml) was injected into OA menisci explants (n=6; female, age 68–74; mean 71±2). Explants were cultured for 2 weeks until analysis. (A) Safranin-O staining of the human meniscus explants showing entire meniscus, vascular (VAS), intermediate (INT), avascular (AVA) and superficial area (SUP). (B) Meniscus histopathological scores. P < 0.05 (One-way ANOVA) compared to the selected group. (C) Immunohistochemistry for MKX, MMP13, COL10A1, and RUNX2. (D) qRT-PCR analysis of genes related to meniscus ECM, degeneration, hypertrophy, inflammation, and calcification. P < 0.05 (Mann-Whitney test) compared to the Ad-GFP injected meniscus group. All values are mean ± s.e.m. Gray scale bars: 1mm; black scale bars: 100 µm.