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Published on: June 22, 2013
Spreading of embryologically distinct urothelial cells is inhibited by SPARC
Amber E Hudson1, Waldo C Feng, Catherine F Delostrinos
1Division of Pediatric Urology, Children's Hospital and Regional Medical Center, Seattle, Washington 98105-0371, USA.
Abstract:
The AON epitope of secreted protein acidic and rich in cysteine (SPARC) is a conserved motif expressed by human SPARC in a variety of human cell types. Through the use of a monoclonal antibody that recognizes this epitope, transitional epithelium was found to restrict expression of SPARC to the suprabasal and intermediate layer. Such intracellular expression was defined by immunoreactive signals that localized to the apical plasma membranes of suprabasal and intermediate cells. Polarization of SPARC to apical plasma membranes of suprabasal cells was retained in vitro by a subpopulation of cells that exhibited characteristics of suprabasal cells--cell-cycle quiescence, large cell volumes, and multiple nuclei. In contrast, the basal layer of transitional epithelium in vivo and cycling cells in vitro did not exhibit this apical staining pattern, but instead sequestered the SPARC polypeptide within urothelial cytoplasm and/or nuclei, as revealed by immunohistochemical analysis. Elution of soluble proteins and DNA from urothelial cells revealed the presence of SPARC within the nuclear matrix--and that SPARC colocalized with the nuclear matrix Ki-67 antigen. rSPARC activity was demonstrated and quantified with a rounding assay whereby the spreading of freshly plated cells was inhibited by recombinant SPARC in a concentration- and time-dependent manner. Inhibition of spreading was observed in urothelial cells derived from endoderm (bladder) and mesoderm (ureter) germ layers. Statistically significant differences were seen between urothelial cells from these two layers. Mesodermal cells recovered more slowly from the inhibitory effects of rSPARC, such that at hour 6 endodermal cells underwent significantly more spreading, as shown by a rounding index (RI). These experiments provide new insights about the matricellular trafficking of SPARC and suggest that intra- and extra-cellular localization patterns influence the development, homeostasis, and differentiation of transitional epithelium.
Insights
Secreted protein acidic and rich in cysteine (SPARC) localization varies in transitional epithelium, impacting cell behavior. SPARC
Area of Science:
- Cell Biology
- Urothelial Biology
- Extracellular Matrix Proteins
Background:
- Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein with diverse roles.
- SPARC expression and localization in transitional epithelium are not fully understood.
- The AON epitope provides a specific target for studying SPARC in urothelial cells.
Purpose of the Study:
- To investigate the precise localization and functional impact of SPARC within transitional epithelium.
- To determine how SPARC's intracellular and extracellular distribution affects urothelial cell behavior.
- To compare SPARC's effects on urothelial cells derived from different germ layers.
Main Methods:
- Immunohistochemical analysis using a monoclonal antibody against the SPARC AON epitope.
- In vitro cell culture of urothelial cells with characterization of suprabasal cell subpopulations.
- Nuclear matrix protein extraction and co-localization studies with Ki-67.
- Recombinant SPARC (rSPARC) activity assay using a cell spreading (rounding) assay.
Main Results:
- SPARC is restricted to suprabasal and intermediate layers of transitional epithelium, localizing to apical plasma membranes.
- Basal and cycling cells sequester SPARC in cytoplasm and/or nuclei, including the nuclear matrix.
- SPARC colocalizes with the nuclear matrix Ki-67 antigen.
- Recombinant SPARC inhibits urothelial cell spreading in a concentration- and time-dependent manner.
- Endodermal (bladder) cells show faster recovery from rSPARC inhibition compared to mesodermal (ureter) cells.
Conclusions:
- SPARC exhibits distinct intracellular and extracellular localization patterns in transitional epithelium.
- SPARC's localization within the nuclear matrix suggests a role in nuclear function.
- The differential response of endodermal and mesodermal urothelial cells to SPARC highlights germ layer-specific effects.
- Intra- and extracellular SPARC localization influences transitional epithelium development, homeostasis, and differentiation.
