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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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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.
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pone.0146124.g003: 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.

Mentions: In one day-cultured pellets we confirmed overexpression of TGFBR1 following caALK5 transduction (Fig 3A) and ACVRL1 overexpression following caALK1 transduction (Fig 3B). Whereas overexpression of caALK1 did not change TGFBR1 expression (Fig 3A), caALK5 overexpression reduced mRNA levels of ACVRL1 (Fig 3B). We also verified that caALK5 and caALK1 receptors phosphorylated their downstream signaling proteins SMAD2/3 (Fig 3C) and SMAD1/5/8 (Fig 3D) without prior activation by TGFβ and dimerization with type II receptors.


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)

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.
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getmorefigures.php?uid=PMC4697836&req=5

pone.0146124.g003: 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.
Mentions: In one day-cultured pellets we confirmed overexpression of TGFBR1 following caALK5 transduction (Fig 3A) and ACVRL1 overexpression following caALK1 transduction (Fig 3B). Whereas overexpression of caALK1 did not change TGFBR1 expression (Fig 3A), caALK5 overexpression reduced mRNA levels of ACVRL1 (Fig 3B). We also verified that caALK5 and caALK1 receptors phosphorylated their downstream signaling proteins SMAD2/3 (Fig 3C) and SMAD1/5/8 (Fig 3D) without prior activation by TGFβ and dimerization with type II receptors.

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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Related in: MedlinePlus