Limits...
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.

Show MeSH

Related in: MedlinePlus

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0146124.g001: 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.

Mentions: Transforming Growth Factor-β is known to signal either via ALK5 or ALK1 receptors, which intracellularly activate SMAD2/3 and SMAD1/5/8 signaling respectively. We first confirmed gene expression of ALK5 (TGFBR1) and ALK1 (ACVRL1) in in vitro-expanded BMSCs (n = 5) and observed that their expression levels varied between donors (Fig 1).


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)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4697836&req=5

pone.0146124.g001: 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.
Mentions: Transforming Growth Factor-β is known to signal either via ALK5 or ALK1 receptors, which intracellularly activate SMAD2/3 and SMAD1/5/8 signaling respectively. We first confirmed gene expression of ALK5 (TGFBR1) and ALK1 (ACVRL1) in in vitro-expanded BMSCs (n = 5) and observed that their expression levels varied between donors (Fig 1).

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