ASPET Poster Materials and Methods and References

Techniques

Work in the lab uses cell culture models of kidney (OK, LLC-PK1 and JTC12 cells), bone (UMR106, Saos-2 and MC3T3 obsteoblasts), fibroblasts (NIH 3T3-L1) and macrophages.  We also use a variety of assays to measure changes in signaling pathway activity including cell-based enzyme-lined immunosorbent assays (ELISA), radioimmunoassay (RIA), protein and nucleic acid analysis by electrophoresis and blotting, assessment of receptor characteristics using ligand binding assays, cell proliferation assays, immunoprecipitation, and  immunofluorescence. We also measure second messenger production using reporters  for cAMP (GloSensor cAMP; below left) and Ca2+ (a fluorogenic calcium-binding dye; below right).

Cell Models

Renal Cells:  OK cells (pictured left) are models of the renal proximal tubules and actively transport fluid and electrolytes from the apical to the basolateral surface.  When they are grown on plastic culture dishes, the transport of fluid actually pushes cells up off the plate, forming "domes".  OK cells are among the few continual cell lines that express high affinity PTH1R (Kd = 1 nM) that regulate Na+-dependent phosphate transport, Na+/K+-ATPase  and Na+/H+ exchange.  Clonal populations (particularly the NHERF-deficient OKH cells) have been used to define the important of PLCβ and Ca2+ signaling in the regulation of Na+-dependent phosphate transport.  In additional, a clone deficient in the scaffolding molecule NHERF1 has been used to define the role of this protein in PLCβ activation and the regulation of Na+/K+-ATPase activity.  We are also using these cells to examine the PTH signaling pathways used by the hormone  to activate ERK.  Other renal cell models used in the lab include JTC12 monkey kidney cells and LLC-PK1 pig kidney cells.  JTC12 cells express PTH receptors and Na+-dependent phosphate transporters, but the transporters are not regulated by PTH.  LLC-PK1 cells do not express PTH receptors but have many features of the proximal nephron.

Bone Cells:  UMR106 Cells are osteoblasts-like osteosarcoma cells that express PTH receptors and are also responsive to PGE2 and bone resorbing steroids.  UMR106 cells have been used to examine the regulation of collagen synthesis in response to a variety of drugs and hormone and use of different concentrations of PTH have uncovered PLC and AC-specific responses in these cells. We also use MC3T3-E1 Cells which are pre-obsteoblasts that can be induced to differentiate by growing them in ascorbic acid and 3-4 mM  inorganic phosphate.  Mineralizing (differentiated) subclones express mRNAs for the osteoblast markers including bone sialoprotein (BSP), osteocalcin and the PTH receptor.  SaOS Cells are osteoblasts-like osteosarcoma cells that produce mineralized matrix under appropriate culture conditions.  We have used SaOS cells to examine the signaling pathways activated by PTH (1-34) and PTH (7-34) and the differences in gene expression profiles produced by each PTH peptide.

Vascular Smooth Muscle Cells (VSMC):  We have used primary cultures of human aortic smooth muscle cells and a murine aortic smooth muscle (MOVAS) cell line to examine the role of α1 adrenergic receptors in the regulation fo VSMC hypertrophy and migration, processes that are activated in hypertension.

IC-21 Macrophages:  These cells are a continual monoclonal murine macrophage-like cell line. IC-21 cells are morphologically indistinguishable from macrophages for the first few days after subculturing and are phagocytic and cytolytic. IC-21 cells express macrophage-specific receptors, including those for IgG2a, IgG2b, complement receptor C3, and platelet-activating factor receptor and respond to macrophage-specific antigens.  We have used the cells to model the effects of the intestinal parasite Giardia lamblia on immune cells signaling.