Hsp47-dependent and -independent intracellular trafficking of type I collagen in corneal endothelial cells.
OBJECTIVE
Type I collagen is post-translationally regulated in corneal endothelial cells (CEC): CEC synthesize procollagen I and degrade it intracellularly. We investigated whether there is a Hsp47-independent pathway during intracellular trafficking of procollagen I.


METHODS
Specific inhibitors were used to block intracellular transport of procollagen I and Hsp47. Immunocytochemical analysis was performed to determine the intracellular localization of the proteins of interest.


RESULTS
When cells were treated with alpha,alpha'-dipyridyl, this specific inhibitor for collagen promoted retention in the endoplasmic reticulum (ER) of some of the underhydroxylated procollagen I, which was colocalized with Hsp47 in CEC. At the same time, another fraction of the alpha,alpha'-dipyridyl-induced underhydroxylated procollagen I was not located in the ER. When CEC were treated with brefeldin A, procollagen I and Hsp47 demonstrated a high degree of colocalization at the ER, whereas the inhibitor had less of an effect on the compartmentalization of procollagen I and prolyl 4-hydroxylase. When CEC were treated with either monensin or bafilomycin A1, procollagen I and Hsp47 were not colocalized: procollagen I was mostly localized at the Golgi area, while Hsp47 predominantly showed ER distribution. When colocalization of procollagen I and prolyl 4-hydroxylase was examined, a major population of procollagen I was not colocalized with prolyl 4-hydroxylase in the ER.


CONCLUSIONS
These results indicate that some procollagen I and Hsp47 travel together from the ER to the cis-Golgi compartment and that a major population of procollagen I that may not be properly hydroxylated may be destroyed intracellularly via the Hsp47-independent pathway in CEC.
Ko
M K
MK
Doheny Eye Institute, University of Southern California School of Medicine, Los Angeles, CA 90033, USA. minheeki@hsc.usc.edu
Kay
E P
EP
D015082:2,2'-Dipyridyl / Q000494:pharmacology; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D020126:Brefeldin A / Q000494:pharmacology; D002478:Cells, Cultured; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D004728:Endothelium, Corneal / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000378:metabolism*; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D018942:Macrolides; D018613:Microscopy, Confocal; D008985:Monensin / Q000494:pharmacology; D011347:Procollagen / Q000378:metabolism*; D011393:Procollagen-Proline Dioxygenase / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011817:Rabbits
D000900:Anti-Bacterial Agents; D004791:Enzyme Inhibitors; D006360:Heat-Shock Proteins; D018942:Macrolides; D011347:Procollagen; D011500:Protein Synthesis Inhibitors; D020126:Brefeldin A; D015082:2,2'-Dipyridyl; C040929:bafilomycin A1; D008985:Monensin; D011393:Procollagen-Proline Dioxygenase