Abstract
CCN5, a member of the CCN family of growth factors, inhibits the proliferation and migration of smooth muscle cells in cell culture and animal models. Expressed in both embryonic and adult tissues, CCN5 exhibits a matricellular localization pattern characteristic of secreted proteins that are closely associated with the cell surface. In addition to this observed expression pattern, immunohistochemical evidence suggests the presence of nuclear CCN5 in some cells. To determine if CCN5 localizes to the nucleus we performed immunofluorescence, confocal imaging, and cell fractionation to corroborate the immunohistochemical observations. After confirming the presence of nuclear CCN5 using four independent experimental methods, we identified a single putative nuclear localization signal in the von Willebrand factor C domain of mouse and rat CCN5. Site directed mutagenesis of the three basic amino acids in the putative nuclear localization sequence did not prevent nuclear localization of CCN5 in four different cell types, suggesting that CCN5 nuclear transport is not mediated by the only canonical nuclear localization signal present in the primary amino acid sequence. Future work will address the mechanism of nuclear localization and the function of nuclear versus secreted CCN5.
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Abbreviations
- (CT):
-
Carboxy terminal domain
- (CCN):
-
cysteine rich 61/connective tissue growth factor/nephroblastoma overexpressed
- (GFP):
-
green fluorescent protein
- (hAoSMC):
-
human aortic smooth muscle cells
- (IGFBP):
-
insulin-like growth factor-binding protein domain
- (NLS):
-
nuclear localization signal(s)
- (SMC):
-
smooth muscle cell
- (TSP-1):
-
thrombospondin-1 domain
- (VSMC):
-
vascular smooth muscle cells
- (VWC):
-
von Willebrand Factor-C domain
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Acknowledgments
This paper is dedicated to the memory of Mark Gray. The work was support in part by grants HD046251 and HL49973 to JJC and an American Heart Association Fellowship to CB.
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Wiesman, K.C., Wei, L., Baughman, C. et al. CCN5, a secreted protein, localizes to the nucleus. J. Cell Commun. Signal. 4, 91–98 (2010). https://doi.org/10.1007/s12079-010-0087-x
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DOI: https://doi.org/10.1007/s12079-010-0087-x