Mammalian suppressor-of-fused modulates nuclear-cytoplasmic shuttling of Gli-1.

Sonic hedgehog, Patched and Gli are components of a mammalian signalling pathway that has been conserved during evolution and which has a central role in the control of pattern formation and cellular proliferation during development. Here we identify the human Suppressor-of-Fused (SUFUH) complementary DNA and show that the gene product interacts physically with the transcriptional effector GLI-1, can sequester GLI-1 in the cytoplasm, but can also interact with GLI-1 on DNA. Functionally, SUFUH inhibits transcriptional activation by GLI-1, as well as osteogenic differentiation in response to signalling from Sonic hedgehog. Localization of GLI-1 is influenced by the presence of a nuclear-export signal, and GLI-1 becomes constitutively nuclear when this signal is mutated or nuclear export is inhibited. These results show that SUFUH is a conserved negative regulator of GLI-1 signalling that may affect nuclear-cytoplasmic shuttling of GLI-1 or the activity of GLI-1 in the nucleus and thereby modulate cellular responses.

Mechanical strain increases protein tyrosine phosphorylation in airway smooth muscle cells.

Mechanical stress contributes to normal structure and function of the lung as well as pathology in such diseases as bronchopulmonary dysplasia and adult respiratory distress syndrome. Stress-related increases in airway smooth muscle (ASM) quantity are reflected in vitro where cultured ASM cells respond to cyclic deformational strain with increased proliferation, cell reorientation, protein production, stress fibers, and focal adhesions. To understand the mechanisms of mechanical signaling in ASM cells, we investigated whether strain increased tyrosine phosphorylation of focal adhesion-related proteins. ASM cells were grown to confluence on collagen type I and subjected to 30 min of cyclic deformation strain (2 s of 25% deformation of the substratum, 2 s relaxation) and compared at various time points with identical cells not subjected to strain for phosphotyrosine content of three focal adhesion-concentrated proteins (pp125FAK, paxillin, and talin) by Western blotting. Strain caused a rapid increase in tyrosine phosphorylation of pp125FAK and paxillin. Tyrosine phosphorylation decreased by 4 h in pp125FAK after discontinuing strain but remained elevated in paxillin at 24 h. Increases in tyrosine phosphorylation of talin were not found. In separate studies, when cells were strained in the presence of tyrosine kinase inhibitors (genistein and herbimycin A), strain-induced reorientation and elongation were inhibited. Mechanochemical signal transduction appears to mediate cell morphologic changes through quantitative and possibly qualitative changes in tyrosine phosphorylation of adhesion-related proteins.

Strain reorganizes focal adhesions and cytoskeleton in cultured airway smooth muscle cells.

Abnormal mechanical stress on pulmonary structures is associated with increased airway resistance and impaired gas exchange as a result of increased airway smooth muscle (ASM) deposition. Using an in vitro system with cultured ASM cells, we have demonstrated that cyclic deformational strain increases ASM cellular myosin and myosin light chain kinase. To determine if these contractile protein increases were accompanied by ultrastructural changes in cells indicating phenotypic modulation, cells subjected to strain were compared to cells grown under static conditions by transmission electron microscopy (TEM) and fluorescent staining. The strained ASM cells oriented perpendicular to the strain direction were more elongated and contained more actin stress fibers than identical cells grown under physically static conditions. The stress fiber bundles were thicker and reorganized parallel to the long axis of the cell. Marked increases in the numbers and lengths of focal adhesions between the cell membrane and the substratum were found by both TEM and immunostaining for talin. Mechanical strain thus increases organization of cytoskeletal elements in cultured ASM cells. Similar effects in vivo may serve to promote the expression of the contractile phenotype of cultured ASM cells independent of other in vivo factors and alter cell contractility. Increased organization of cytoskeletal elements might also increase the efficiency of signal transduction from the extracellular matrix into the cell interior.