Regulated expression of the Anaplastic Lymphoma Kinase (ALK) in cultured rat hepatic stellate cells during transdifferentiation into myofibroblasts

Abstract

Anaplastic Lymphoma Kinase (ALK) has been identified as a receptor tyrosine kinase for the growth factor pleiotrophin. ALK belongs to the insulin-receptor related receptor tyrosine kinases and till now ALK expression was exclusively described at selected sites in the nervous system. Neurotrophic factors and their receptors are related in non-neural tissue to differentiation and tissue remodelling after injury. We found ALK to be expressed on isolated primary rat hepatic stellate cells (HSCs) and examined whether in vitro transdifferentiation of HSCs into myofibroblasts may be associated with changes in ALK expression. Semi-quantitative RT-PCR analysis revealed that ALK mRNA could be detected in low levels in most liver parenchymal and non-parenchymal cells suggesting that ALK expression is more widely spread as expected from published data. However, the highest transcription levels have been detected in quiescent HSCs and in vitro transdifferentiation caused down-regulation of ALK expression in myofibroblasts. Immunofluorescence staining revealed a prominent localisation of the ALK tyrosine receptor in focal membrane areas of quiescent HSC. Cell lysates from purified primary rat HSC and from the HS cell line HSC-T6 were subjected to immunoprecipitation and subsequent Western blotting with anti-ALK antibodies. This approach revealed a prominent 200 kDa band corresponding to the ALK glycoprotein in both extracts. Pleiotrophin (PTN), a heparin-binding protein and a growth factor with a broad spectrum of biological activities, has recently been identified as a ligand for ALK. Northern blot analysis of PTN mRNA expression demonstrated a very similar down-regulation of the ligand during in vitro transdifferentiation of HSC suggesting that PTN may signal via ALK in an autocrine mode and both, receptor and its ligand are associated with the quiescent stage of HSC.