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The Laboratory of Dr. James Bassuk

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PROJECTS of the Program in Human Urothelial Biology

Regulators of Human Urothelial Cell Proliferation

The overall goal is to develop recombinant forms of these regulators for use in the treatment of urinary tract disorders in children and adults. Our laboratory is well funded by multiple NIH grants and is staffed by physician scientists, basic scientists, fellows, residents, postdoctoral trainees, technicians, and students. We seek to better understand how the urothelium grows, differentiates, and responds to injury.

Background
The urothelial cells that line the renal pelvis, ureter, bladder, and parts of the urethra interact with each other and with their basement membrane to form transitional epithelium, a dynamic structure that displays a remarkable ability to proliferate and change its shape. Although metabolically active, normal urothelial cells rest in the G0 phase of the cell cycle for approximately one year -- longer than any other epithelia of the body. In response to external stimuli, normal bladder urothelial cells will exit from G0, traverse the cell cycle, and express proliferation markers within 24-48 hours. The balance between positive and negative factors that control urothelial cell proliferation is an area that my laboratory is particularly interested in. Such balance is influenced by signals from cells, the extracellular matrix, and the urothelial basement membrane. Additional signals arise from the urinary response to injury or from urinary carcinogens derived from exposure to tobacco and wood smoke, diesel exhaust, and environmental or occupational toxins.

The following projects are based on new and innovative methods designed to strengthen our translational approach to the problem of bladder and urinary tract disease.

Positive Signals that Promote Urothelial Proliferation
FGF-10 is a small, basic polypeptide required for proper development of the urinary tract. It plays an important role in regulating DNA synthesis of urothelial cells, a crucial process involved in the control of growth, differentiation, and repair of the urothelium. We are specifically interested in learning about the mode of action for FGF-10.

·   Development of recombinant (r) FGF-10 as a tool to study urothelial cell biology. The
     technology of generating biologically active rFGF-10 is well established in our laboratory.

·   Induction of urothelial cell proliferation by rFGF-10. Two pathways have been identified in
     primary cultures of human cells:

     a) Activation of a novel isoform of the FGFR2IIIb surface receptor that involves Tyr
           phosphorylation and heparin

     b) Translocation of rFGF-10 into nuclei. We intend to identify the nuclear macromolecules
           that interact with rFGF-10 to control progression through the cell cycle.

·   The paracrine mode of action of FGF-10. This process is described by a complex network of
     action that originates in the mesenchyme (fibroblasts of the lamina propria) but acts on the
     urothelium. In situ hybridization and complex cell cultures systems are being used to
     delineate the precise mechanisms.

Negative Signals that Inhibit Proliferation
SPARC is a small, acidic hydrophobic glycoprotein originally described as osteonectin, an abundant protein of bone. Many cell types (normal and transformed) and tissues of diverse embryonic origin express and secrete the product of this single-copy gene.

SPARC is a matricellular protein that interacts with growth factors, the extracellular matrix, glomerular cells of the mesangium and visceral epithelium, and urothelial cells of transitional epithelium. Intra- and extra- cellular localization patterns of SPARC are have been linked to the development, homeostasis, and differentiation of transitional epithelium. The protein has the ability to travel in and out of urothelial cells and to undergo translocation into urothelial cell nuclei.

Specific projects are underway to better understand how SPARC functions in the context of a dynamic steady-state interrelationship that suppresses the progression of the urothelial cell cycle and mediates the attachment of urothelial cells to its underlying basement membrane.

·    Development of recombinant (r) SPARC as a tool to study urothelial cell biology. The
      technology of generating biologically active rSPARC is well established in our laboratory.
      Because rSPARC is expressed in an insoluble form, the renaturation process in itself has
      proved to be a model system of how proteins fold in solution. These efforts have used a
      variety of biophysical strategies. Formulation efforts are underway to optimize the
      stabilization of the protein for long term storage.

Evaluation of Heterogeneity of Urothelial Cells in Interstitial Cystitis

A new initiative is underway to determine the viability and cell-subtype profiles of urothelial cells exfoliated into urine. Profiles from normal controls will be compared to profiles of subjects that present with pelvic pain and interstitial cystitis. These studies are designed to understand the mechanisms involved in converting the transitional epithelium from a quiescent to active or changed tissue. Such changes are hypothesized to be integral to pelvic pain, urinary frequency and urgency.

This Web Page was Last Edited on 05/31/05