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 p olypeptide that promotes proliferation, collagen synthesis, and chemotaxis in mesanchymal cells. When coinjected subcutaneously with transforming grow th factor beta (TGF beta), CTGF promotes sustained fibrosis in rats. Howeve r, little is known about the cell biology and structure/functional relation ship of CTGF. In particular, no detailed characterization of the subcellula r localization of CTGF has occurred, nor have sequences been identified wit hin this protein required for this localization. In this report, using immu nofluorescence and Western blot analysis, we show that CTGF is localized to the Golgi apparatus both in dermal fibroblasts and activated hepatic stell ate cells. Using these methods, no CTGF was detected in endosomal, plasma m embrane, cytosolic or nuclear fractions. Addition of brefeldin A, a drug th at disrupts the Golgi, blocks the secretion of CTGF. We further show that t he amino-terminal 37 amino acids of CTGF are sufficient to localize a heter ologous protein (red fluorescent protein, RFP) to the Golgi. Although withi n this region of human CTGF isa N-glycosylation site, tunicamycin, which bl ocks N-linked glycosylation, has no significant effect on CTGF secretion. S urprisingly, mutation of a single amino acid residue, CYS-34, to alanine pr events localization of a CTGF-RFP fusion protein to the Golgi. These result s are the first proof that endogenous CTGF is localized to the Golgi appara tus. Furthermore, using exogenously added I-125-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. (C) 2001 Academic Press.