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Matricellular protein SPARC is translocated to the nuclei of immortalized murine lens epithelial cells
Qi Yan1, Matt Weaver, Nikole Perdue
1Hope Heart Program, Benaroya Research Institute at Virginia Mason, Seattle, Washington 98104-2046, USA. qyan@hopeheart.org
Abstract:
The matricellular glycoprotein, secreted protein acidic and rich in cysteine (SPARC), has complex biological activities and is important for lens epithelial cell function and regulation of cataract formation. To understand how SPARC influences lens epithelial cell activity and homeostasis, we have studied the subcellular distribution of SPARC in murine lens epithelial cells in vitro. We demonstrate that endogenous SPARC is located in the cytoplasm of either quiescent or dividing lens epithelial cells in culture. However, cytoplasmic SPARC was translocated into the nuclei of immortalized lens epithelial cells upon a significant reduction of intracellular SPARC in these cells. Recombinant human (rh) SPARC added to the culture media was quickly and efficiently internalized into the cytosol of SPARC-null lens epithelial cells. Moreover, cytoplasmic rhSPARC was also translocated into the nucleus after exogenous rhSPARC was removed from the culture media. The translocation of SPARC into the nucleus was therefore triggered by the reduction of SPARC protein normally available to the cells. A mouse SPARC-EGFP chimeric fusion protein (70 kDa) was expressed in lens epithelial cells and 293-EBNA cells, and was observed both in the cytoplasm and culture medium, but not in the nucleus. SPARC does not appear to have a strong nuclear localization sequence. Alternatively, SPARC might pass through the nuclear pore complex by passive diffusion. SPARC therefore functions not only as an extracellular protein but also potentially as an intracellular protein to influence cellular activities and homeostasis.
Insights
Secreted protein acidic and rich in cysteine (SPARC) is found in the cytoplasm of lens cells but translocates to the nucleus when intracellular levels decrease. This suggests SPARC has intracellular functions influencing cell homeostasis.
Area of Science:
- Ophthalmology
- Cell Biology
- Biochemistry
Background:
- Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein crucial for lens epithelial cell function and cataract regulation.
- The precise role and localization of SPARC within lens epithelial cells remain incompletely understood.
Purpose of the Study:
- To investigate the subcellular distribution of SPARC in murine lens epithelial cells.
- To determine how SPARC influences lens epithelial cell activity and homeostasis.
Main Methods:
- Studied the subcellular localization of endogenous SPARC in cultured murine lens epithelial cells (quiescent and dividing).
- Examined SPARC translocation in immortalized lens epithelial cells with reduced intracellular SPARC.
- Investigated uptake and translocation of recombinant human (rh) SPARC in SPARC-null lens epithelial cells.
- Expressed a SPARC-EGFP fusion protein in lens epithelial cells and 293-EBNA cells.
Main Results:
- Endogenous SPARC is primarily located in the cytoplasm of quiescent and dividing lens epithelial cells.
- Cytoplasmic SPARC translocated to the nucleus in immortalized cells when intracellular SPARC levels were reduced.
- Exogenous rhSPARC was internalized into the cytosol and subsequently translocated to the nucleus after removal from the culture medium.
- A SPARC-EGFP fusion protein was found in the cytoplasm and medium, but not the nucleus, suggesting SPARC may enter the nucleus via passive diffusion.
Conclusions:
- SPARC functions as both an extracellular and intracellular protein.
- Reduction in cellular SPARC levels triggers its translocation into the nucleus.
- SPARC plays a role in intracellular processes influencing cellular activities and homeostasis in lens epithelial cells.
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