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Objectives. Urinary stem cells (USCs) have the capacity for unlimited growth and are promising tools for the investigations of cell differentiation and urinary regeneration. However, the limited life span significantly restricts their usefulness. This study is aimed at exploring the effect of integrin-linked kinase (ILK) on the smooth muscle cells (SMCs) differentiation of the dog USCs and investigating its molecular mechanism. Methods. An immortalized USCs cell line with the molecular markers and biological functions was prepared. After successfully inducing the differentiation of USCs into SMCs, the expression level of the unique key factor and its mechanisms in this process was determined through real-time polymerase chain reaction, Western blot, or Immunofluorescence staining. Results. We found that high cell density promoted USCs differentiation SMCs, and ILK was necessary for USCs differentiation into SMCs. Knocking down ILK decreased the expression of SMCs specific-marker, while using a selective ILK agonist increased the expression of SMCs specific-marker. Furthermore, ILK regulated SMCs differentiation in part through the activation of NF-κB pathway in USCs. A NF-κB activity assay showed overexpression of ILK could significantly upregulate NF-κB p50 expression, and NF-κB p50 acts as downstream signal molecular of ILK. Conclusion. High cell density induces the differentiation of USCs into SMCs, and ILK is a key regulator of myogenesis. Furthermore, NF-κB signaling pathway might play a crucial role in this process.

Objectives Urinary stem cells (USCs) have the capacity for unlimited growth and are promising tools for the investigations of cell differentiation and urinary regeneration. However, the limited life span significantly restricts their usefulness. This study is aimed at exploring the effect of integrin-linked kinase (ILK) on the smooth muscle cells (SMCs) differentiation of the dog USCs and investigating its molecular mechanism. Methods An immortalized USCs cell line with the molecular markers and biological functions was prepared. After successfully inducing the differentiation of USCs into SMCs, the expression level of the unique key factor and its mechanisms in this process was determined through real-time polymerase chain reaction, Western blot, or Immunofluorescence staining. Results We found that high cell density promoted USCs differentiation SMCs, and ILK was necessary for USCs differentiation into SMCs. Knocking down ILK decreased the expression of SMCs specific-marker, while using a selective ILK agonist increased the expression of SMCs specific-marker. Furthermore, ILK regulated SMCs differentiation in part through the activation of NF-κB pathway in USCs. A NF-κB activity assay showed overexpression of ILK could significantly upregulate NF-κB p50 expression, and NF-κB p50 acts as downstream signal molecular of ILK. Conclusion High cell density induces the differentiation of USCs into SMCs, and ILK is a key regulator of myogenesis. Furthermore, NF-κB signaling pathway might play a crucial role in this process.