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

de Kroon LM, Narcisi R, Blaney Davidson EN, Cleary MA, van Beuningen HM, Koevoet WJ, van Osch GJ, van der Kraan PM - PLoS ONE (2015)

Bottom Line: 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.Moreover, short hairpin-mediated downregulation of either ALK5 or ALK1 resulted in a strong inhibition of TGFβ-induced chondrogenesis.These results imply that optimizing cartilage formation by mesenchymal stem cells will depend on activation of both receptors.

View Article: PubMed Central - PubMed

Affiliation: Department of Rheumatology, Experimental Rheumatology, Radboud University Medical Center, Nijmegen, the Netherlands.

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.

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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.
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pone.0146124.g005: 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.

Mentions: At day 1 and 7 of chondrogenic induction we verified the efficiency of shRNA-mediated downregulation by gene expression analysis of TGFBR1 and ACVRL1. Compared to the control condition at both time points, TGFBR1 expression was about 50% downregulated by ALK5-shRNA (Fig 5A) and ACVRL1 expression was about 50% downregulated by ALK1-shRNA (Fig 5B). In addition, TGFBR1 mRNA levels were not affected by ALK1-shRNA (Fig 5A), but ACVRL1 was higher expressed in the ALK5-shRNA condition than in the control condition at day 7 (Fig 5B).The effect of shRNA-mediated ALK5 and ALK1 downregulation on TGFβ-induced chondrogenic differentiation of BMSCs was determined by ACAN and COL2A1 gene expression analysis and examining deposition of proteoglycans and collagen type II in pellets stimulated with TGFβ for 7 days. Compared to control vector-transduced pellets stimulated with TGFβ, ALK5-shRNA reduced expression levels of ACAN by 98.8% (Fig 5C) and COL2A1 by 99.9% (Fig 5D), resulting in the same low ACAN and COL2A1 mRNA levels as in the unstimulated control condition. ALK1-shRNA reduced ACAN expression by 62.6% (Fig 5C) and COL2A1 expression by 87.2% (Fig 5D) compared with the TGFβ-stimulated control condition. Furthermore, we observed that control vector-transduced pellets exposed to TGFβ stained strongly positive for proteoglycans (Fig 5E; upper panel) and collagen type II (Fig 5E; lower panel), whereas TGFβ-stimulated pellets overexpressing ALK1-shRNA were only weakly positive for both cartilage matrix molecules. Moreover, pellets consisting of ALK5-shRNA-transduced BMSCs did not stain positive for proteoglycans nor for collagen type II, despite stimulation with TGFβ (Fig 5E). Thus, shRNA-mediated downregulation of ALK5 or ALK1 results in an abrogation of TGFβ-induced chondrogenic differentiation of BMSCs.


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

de Kroon LM, Narcisi R, Blaney Davidson EN, Cleary MA, van Beuningen HM, Koevoet WJ, van Osch GJ, van der Kraan PM - PLoS ONE (2015)

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.
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Related In: Results  -  Collection

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pone.0146124.g005: 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.
Mentions: At day 1 and 7 of chondrogenic induction we verified the efficiency of shRNA-mediated downregulation by gene expression analysis of TGFBR1 and ACVRL1. Compared to the control condition at both time points, TGFBR1 expression was about 50% downregulated by ALK5-shRNA (Fig 5A) and ACVRL1 expression was about 50% downregulated by ALK1-shRNA (Fig 5B). In addition, TGFBR1 mRNA levels were not affected by ALK1-shRNA (Fig 5A), but ACVRL1 was higher expressed in the ALK5-shRNA condition than in the control condition at day 7 (Fig 5B).The effect of shRNA-mediated ALK5 and ALK1 downregulation on TGFβ-induced chondrogenic differentiation of BMSCs was determined by ACAN and COL2A1 gene expression analysis and examining deposition of proteoglycans and collagen type II in pellets stimulated with TGFβ for 7 days. Compared to control vector-transduced pellets stimulated with TGFβ, ALK5-shRNA reduced expression levels of ACAN by 98.8% (Fig 5C) and COL2A1 by 99.9% (Fig 5D), resulting in the same low ACAN and COL2A1 mRNA levels as in the unstimulated control condition. ALK1-shRNA reduced ACAN expression by 62.6% (Fig 5C) and COL2A1 expression by 87.2% (Fig 5D) compared with the TGFβ-stimulated control condition. Furthermore, we observed that control vector-transduced pellets exposed to TGFβ stained strongly positive for proteoglycans (Fig 5E; upper panel) and collagen type II (Fig 5E; lower panel), whereas TGFβ-stimulated pellets overexpressing ALK1-shRNA were only weakly positive for both cartilage matrix molecules. Moreover, pellets consisting of ALK5-shRNA-transduced BMSCs did not stain positive for proteoglycans nor for collagen type II, despite stimulation with TGFβ (Fig 5E). Thus, shRNA-mediated downregulation of ALK5 or ALK1 results in an abrogation of TGFβ-induced chondrogenic differentiation of BMSCs.

Bottom Line: 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.Moreover, short hairpin-mediated downregulation of either ALK5 or ALK1 resulted in a strong inhibition of TGFβ-induced chondrogenesis.These results imply that optimizing cartilage formation by mesenchymal stem cells will depend on activation of both receptors.

View Article: PubMed Central - PubMed

Affiliation: Department of Rheumatology, Experimental Rheumatology, Radboud University Medical Center, Nijmegen, the Netherlands.

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.

Show MeSH
Related in: MedlinePlus