Connective tissue growth factor is secreted through the Golgi and is degraded in the endosome.
Connective tissue growth factor (CTGF) is a cysteine-rich heparin-binding polypeptide that promotes proliferation, collagen synthesis, and chemotaxis in mesanchymal cells. When coinjected subcutaneously with transforming growth factor beta (TGFbeta), CTGF promotes sustained fibrosis in rats. However, little is known about the cell biology and structure/functional relationship of CTGF. In particular, no detailed characterization of the subcellular localization of CTGF has occurred, nor have sequences been identified within this protein required for this localization. In this report, using immunofluorescence and Western blot analysis, we show that CTGF is localized to the Golgi apparatus both in dermal fibroblasts and activated hepatic stellate cells. Using these methods, no CTGF was detected in endosomal, plasma membrane, cytosolic or nuclear fractions. Addition of brefeldin A, a drug that disrupts the Golgi, blocks the secretion of CTGF. We further show that the amino-terminal 37 amino acids of CTGF are sufficient to localize a heterologous protein (red fluorescent protein, RFP) to the Golgi. Although within this region of human CTGF is a N-glycosylation site, tunicamycin, which blocks N-linked glycosylation, has no significant effect on CTGF secretion. Surprisingly, mutation of a single amino acid residue, CYS-34, to alanine prevents localization of a CTGF-RFP fusion protein to the Golgi. These results are the first proof that endogenous CTGF is localized to the Golgi apparatus. Furthermore, using exogenously added (125)I-labeled CTGF, we show that CTGF is internalized and rapidly degraded in the endosome. That is, CTGF is quantitatively secreted through the golgi and is degraded in the endosome.
Chen
Y
Y
FibroGen, Inc., 225 Gateway Boulevard, South San Francisco, California 94080, USA.
Segarini
P
P
Raoufi
F
F
Bradham
D
D
Leask
A
A
D000818:Animals; D020126:Brefeldin A / Q000494:pharmacology; D002458:Cell Fractionation; D002478:Cells, Cultured; D055513:Connective Tissue Growth Factor; D011992:Endosomes / Q000378:metabolism*; D000078404:Epidermal Cells; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D006056:Golgi Apparatus / Q000378:metabolism*; D006133:Growth Substances / Q000378:metabolism*; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism*; D007150:Immunohistochemistry; D036341:Intercellular Signaling Peptides and Proteins; D008099:Liver / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D000097573:Red Fluorescent Protein
D020126:Brefeldin A; D055513:Connective Tissue Growth Factor; D006133:Growth Substances; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D011500:Protein Synthesis Inhibitors; D011993:Recombinant Fusion Proteins; D016212:Transforming Growth Factor beta; D000097573:Red Fluorescent Protein; C526317:CCN2 protein, human; C526316:CCN2 protein, rat