Activin Receptor-Like Kinase Receptors ALK5 and ALK1 Are Both Required for TGFβ-Induced Chondrogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

Abstract

Introduction: Bone marrow-derived mesenchymal stem cells (BMSCs) are promising for cartilage regeneration because BMSCs can differentiate into cartilage tissue-producing chondrocytes. Transforming Growth Factor β (TGFβ) is crucial for inducing chondrogenic differentiation of BMSCs and is known to signal via Activin receptor-Like Kinase (ALK) receptors ALK5 and ALK1. Since the specific role of these two TGFβ receptors in chondrogenesis is unknown, we investigated whether ALK5 and ALK1 are expressed in BMSCs and whether both receptors are required for chondrogenic differentiation of BMSCs.

Materials & methods: ALK5 and ALK1 gene expression in human BMSCs was determined with RT-qPCR. To induce chondrogenesis, human BMSCs were pellet-cultured in serum-free chondrogenic medium containing TGFβ1. Chondrogenesis was evaluated by aggrecan and collagen type IIα1 RT-qPCR analysis, and histological stainings of proteoglycans and collagen type II. To overexpress constitutively active (ca) receptors, BMSCs were transduced either with caALK5 or caALK1. Expression of ALK5 and ALK1 was downregulated by transducing BMSCs with shRNA against ALK5 or ALK1.

Results: ALK5 and ALK1 were expressed in in vitro-expanded as well as in pellet-cultured BMSCs from five donors, but mRNA levels of both TGFβ receptors did not clearly associate with chondrogenic induction. TGFβ increased ALK5 and decreased ALK1 gene expression in chondrogenically differentiating BMSC pellets. Neither caALK5 nor caALK1 overexpression induced cartilage matrix formation as efficient as that induced by TGFβ. Moreover, short hairpin-mediated downregulation of either ALK5 or ALK1 resulted in a strong inhibition of TGFβ-induced chondrogenesis.

Conclusion: ALK5 as well as ALK1 are required for TGFβ-induced chondrogenic differentiation of BMSCs, and TGFβ not only directly induces chondrogenesis, but also modulates ALK5 and ALK1 receptor signaling in BMSCs. These results imply that optimizing cartilage formation by mesenchymal stem cells will depend on activation of both receptors.

Fig 1. ALK5 and ALK1 are expressed in human bone marrow-derived mesenchymal stem cells (BMSCs).

In human BMSCs, obtained from four adult donors (A1 –A4) and one fetal donor (F1), gene expression levels of ALK5 (= TGFBR1) (A) and ALK1 (= ACVRL1) (B) were analyzed using RT-qPCR. Gene expression was normalized to reference gene RPS27a. Each data point represents one measurement.

Fig 2. Gene expression levels of ALK5 (TGFBR1) and ALK1 (ACVRL1) are not associated with the chondrogenic capacity of human BMSCs.

To induce chondrogenesis, BMSCs (passage 2–4) from adult donors A1 –A5 and fetal donor F1 were pellet-cultured in chondrogenic medium and stimulated with TGFβ. Cartilage-specific gene expression of aggrecan (ACAN) (A) and collagen type IIα1 (COL2A1) (B) was determined in BMSCs cultured for 0, 1, 4, 7 and 21 days using RT-qPCR. Cartilage-specific protein depositions of proteoglycans (C; upper panel) and collagen type II (C; lower panel) were stained in sections of 21 days-cultured pellets. To stain proteoglycans, sections were stained with Safranin O (red/orange) and Fast Green (blue) was used as counterstaining. Collagen type II was stained immunohistochemically (pink) and hematoxylin (purple) was used as counterstaining. Representative images of consecutive pellet sections per donor are shown and the scale bar represents 500 μm. TGFBR1 (D) and ACVRL1 (E) expression levels were measured in BMSC monolayer (day 0) and pellets cultured for 1, 4 and 7 days. Gene expression was normalized to reference gene RPS27a. Data points represent mean ± SD of triplicate BMSC pellets per donor per time point.

Fig 3. BMSCs overexpressing either constitutively active (ca) caALK5 or caALK1 express enhanced levels of phosphorylated SMAD2/3 and SMAD1/5/8 proteins, respectively.

Human fetal BMSCs (donor F1) were transduced with adenoviral caALK5, caALK1 or LacZ as control and pellet-cultured in chondrogenic medium for 1 day. Transduction efficiency of caALK5 and caALK1 was evaluated after gene expression analysis of TGFBR1 (A) and ACVRL1 (B). Gene expression was normalized to reference gene RPS27a and data are expressed as fold change relative to normalized gene levels in LacZ-transduced BMSCs. Bars represent mean ± SD from triplicate pellets of 1 representative experiment (out of 3), **p<0.01; ***p<0.001. Constitutively active receptor signaling of ALK5 and ALK1 was evaluated by Western blot analyses of phosphorylated SMAD (pSMAD) proteins; pSMAD2/3 (C) and pSMAD1/5/8 (D) using GAPDH as loading control.

Fig 4. Constitutively active (ca) caALK5 and caALK1 receptors do not induce BMSC chondrogenesis as efficiently as TGFβ does.

Human fetal BMSCs (donor F1) transduced with adenoviral caALK5, caALK1 or LacZ as control were pellet-cultured in chondrogenic medium for 7 days. Only LacZ-transduced pellets were exposed to TGFβ to compare if either caALK5 or caALK1 induced chondrogenesis as efficiently as induced by TGFβ. Chondrogenesis was evaluated by cartilage-specific gene expression analysis of ACAN (A) and COL2A1 (B) and by staining proteoglycans with Safranin O/Fast Green (C; upper panel) and collagen type II with immunohistochemistry (C; lower panel). Representative images of consecutive pellet sections per condition are shown and the scale bar represents 500 μm. Gene expression was normalized to reference gene RPS27a and data are expressed as % relative to normalized gene levels in LacZ-transduced pellets stimulated with TGFβ. Bars represent mean ± SD from quadruplet pellets of 1 representative experiment (out of 3), ***p<0.001.

Fig 5. shRNA-mediated downregulation of either ALK5 or ALK1 inhibits TGFβ-induced chondrogenic differentiation of BMSCs.

Human fetal BMSCs (donor F1) were transduced with lentiviral ALK5-shRNA, ALK1-shRNA or empty vector as control (Ctrl vector) and pellet-cultured in chondrogenic medium containing TGFβ. Short hairpin RNA-mediated downregulation of ALK5 and ALK1 was determined by gene expression of TGFBR1 (A) and ACVRL1 (B) in pellets cultured for 1 or 7 days. The effect of ALK5-shRNA and ALK1-shRNA on TGFβ-induced chondrogenesis was evaluated by transcript analysis of ACAN (C) and COL2A1 (D) and by staining proteoglycans with Safranin O/Fast Green (E; upper panel) and collagen type II with immunohistochemistry (E; lower panel) in pellets cultured for 7 days. Representative images of consecutive pellet sections per condition are shown and the scale bar represents 500 μm. Gene expression was normalized to reference gene RPS27a and data are expressed as % relative to normalized mRNA levels in control vector-transduced pellets stimulated with TGFβ. Bars represent mean ± SD of quadruplet pellets from 1 representative experiment (out of 3), ns = not significant; **p<0.01; ***p<0.001.