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.