X Gu1, M K Ko, E P Kay. 1. Doheny Eye Institute and the University of Southern California School of Medicine, Los Angeles 90033, USA.
Abstract
PURPOSE: Previous studies by the current investigators showed that type I collagen was posttranslationally regulated in corneal endothelial cells (CECs). These cells synthesize type I procollagen and degrade it intracellularly; however, when CECs are modulated with fibroblast growth factor-2 and/or corneal endothelium modulation factor, they synthesize and secrete type I collagen. Heat shock protein 47 (Hsp47), an endoplasmic reticulum resident protein, is known to function as a molecular chaperon in regulating procollagen folding and/or assembly. The interaction of Hsp47 with type I procollagen synthesis in CECs was also studied. METHODS: Expression of proteins was analyzed by radioactive labeling or immunoblot analysis. The steady state level of type I collagen and Hsp47 mRNAs was determined by northern blot analysis. Coprecipitation using immunoprecipitation followed by immunoblotting was performed to determine the association profile between Hsp47 and type I procollagen. Subcellular localization of Hsp47 and type I procollagen was determined by immunofluorescent staining. RESULTS: Normal and modulated cells expressed Hsp47 and Hsp70. The relative amount of Hsp47 produced by modulated cells was much higher than that of control cells, but the expression level of Hsp70 was the same in control and modulated cells. The steady state levels of type I collagen transcripts were higher in normal cells than in modulated cells, whereas modulated cells contained a much higher steady state level of Hsp47 mRNA. Type I procollagen was found to be associated with Hsp47 when analyzed by coprecipitation or cross-linking. The cytoplasmic localization profile of Hsp47 and type I collagen was different in normal cells, although a colocalization profile was observed to some degree. These two proteins were predominantly colocalized in the Golgi area in modulated CECs. CONCLUSIONS: Hsp47 may be involved in the synthesis and/or intracellular transport of type I collagen in CECs. Modulated cells that secrete type I collagen into the extracellular matrix express a higher level of Hsp47 than do control cells.
PURPOSE: Previous studies by the current investigators showed that type I collagen was posttranslationally regulated in corneal endothelial cells (CECs). These cells synthesize type I procollagen and degrade it intracellularly; however, when CECs are modulated with fibroblast growth factor-2 and/or corneal endothelium modulation factor, they synthesize and secrete type I collagen. Heat shock protein 47 (Hsp47), an endoplasmic reticulum resident protein, is known to function as a molecular chaperon in regulating procollagen folding and/or assembly. The interaction of Hsp47 with type I procollagen synthesis in CECs was also studied. METHODS: Expression of proteins was analyzed by radioactive labeling or immunoblot analysis. The steady state level of type I collagen and Hsp47 mRNAs was determined by northern blot analysis. Coprecipitation using immunoprecipitation followed by immunoblotting was performed to determine the association profile between Hsp47 and type I procollagen. Subcellular localization of Hsp47 and type I procollagen was determined by immunofluorescent staining. RESULTS: Normal and modulated cells expressed Hsp47 and Hsp70. The relative amount of Hsp47 produced by modulated cells was much higher than that of control cells, but the expression level of Hsp70 was the same in control and modulated cells. The steady state levels of type I collagen transcripts were higher in normal cells than in modulated cells, whereas modulated cells contained a much higher steady state level of Hsp47 mRNA. Type I procollagen was found to be associated with Hsp47 when analyzed by coprecipitation or cross-linking. The cytoplasmic localization profile of Hsp47 and type I collagen was different in normal cells, although a colocalization profile was observed to some degree. These two proteins were predominantly colocalized in the Golgi area in modulated CECs. CONCLUSIONS:Hsp47 may be involved in the synthesis and/or intracellular transport of type I collagen in CECs. Modulated cells that secrete type I collagen into the extracellular matrix express a higher level of Hsp47 than do control cells.