Mechanotransduction by endothelial cells is locally generated, direction-dependent, and ligand-specific.

Vascular endothelial cells display a wide panel of responses to changes in the shear stress that is exerted on them by blood flow. How sensory mechanisms convey information about flow conditions and how this information is integrated remains poorly understood. The issue is confounded by: (1) a large number of potential force sensors, (2) difficulties in differentiating these sensors from downstream sites of signal integration, and (3) the complexities inherent in understanding how forces are transmitted from the apical surface of the cell via the cytoskeleton to intracellular sites. As a consequence, neither the structures that sense force nor the nature of the forces that loads them have been clearly defined. In this study, we employed magnetic microspheres coated with ligands that bind integrin subsets (RGD peptides or type I collagen) or PECAM-1 to discriminate the downstream signaling effects of different sensor molecules and mechanisms for how they are loaded. We found that application of force to these transmembrane molecules elicited biologically important signaling (ERK1/2, AKT, and GSK-3beta phosphorylation), and downstream biological responses that depended on the following two factors: (1) the ligand that transmitted force and (2) the direction in which force was applied. These findings indicate that ligands locally generate different shear-induced responses in endothelium that depend on how force is delivered.

Role of Pinin in neural crest, dorsal dermis, and axial skeleton development and its involvement in the regulation of Tcf/Lef activity in mice.

Previous in vitro studies have indicated multiple and varied roles of Pinin (PNN); however, its in vivo role has remained unclear. Here, we report generation of null, hypomorphic, and conditional Pnn alleles in mice. We found that insertion of neomycin-resistance cassette into intron 8 of Pnn resulted in knockdown of Pnn, which allowed Pnn hypomorphic embryos to pass peri-implantation lethality. These mice are lethal at perinatal stages and exhibit defects in the cardiac outflow tract, palate, dorsal dermis, and axial skeleton. Since Wnt/beta-catenin signaling has been shown to play pivotal roles in development of all tissues affected by Pnn hypomorphism, we speculated that Pnn may affect Wnt/beta-catenin signaling. Supporting this view, we demonstrate abnormal activities of Tcf/Lef transcription factors, and alterations in beta-catenin level in multiple Pnn hypomorphic tissues. Taken together, the data suggest that Pnn plays important roles during mouse development through its involvement in regulation of Tcf/Lef activity.

Shear stress regulates forward and reverse planar cell polarity of vascular endothelium in vivo and in vitro.

Cultured vascular endothelium displays profound morphological adaptations to shear stress that include planar cell polarity (PCP) that is directed downstream. Endothelial cells in blood vessels are also polarized; however, the direction of polarity is vessel specific, and shear-independent mechanisms have been inferred. The regulation of endothelial PCP is therefore controversial. We report that the direction of PCP in blood vessels is age and vessel specific; nonetheless, it is caused by shear-related regulation of glycogen synthase kinase-3beta (GSK-3beta), a profound regulator of endothelial microtubule stability. When GSK-3beta is inhibited, PCP reverses direction. Endothelium is the only cell type studied to date that can reverse direction of polarity. Tight regulation of GSK-3beta, microtubule dynamics, and cell polarity was also required for the striking morphological responses of endothelium to shear stress (cell elongation and orientation with shear). Finally, the cytoskeletal polarity displayed in blood vessels is associated with polarized (shear-directed) cell mitoses that have important effects on endothelial repair. Vascular endothelium therefore displays a novel mode of mechanosensitive PCP that represents the first example of a single cell type that can reverse direction of polarity.

Reduction of Pnn by RNAi induces loss of cell-cell adhesion between human corneal epithelial cells.

PURPOSE: Pinin (Pnn/DRS/memA) plays an important role in regulating cell-cell adhesion of corneal epithelial cells. In the nucleus, Pnn interacts with both transcriptional repressor and pre-mRNA processing machinery. Here we investigated the consequences of "knocking down" Pnn expression with short hairpin RNAi (shRNAi) on the corneal epithelial cell phenotype. METHODS: Cultured human corneal epithelial (HCE-T) cells were cotransfected with a shRNAi-expressing construct containing an inverted repeat of a Pnn specific 21 nucleotide sequence (Pnn shRNAi) and a GFP vector as a marker of transfected cells. After 24-48 h, cells were fixed and immunostained with antibodies against Pnn, keratin, desmoplakin, desmoglein, E-cadherin, ZO-1, SR-proteins, and SRm300. To demonstrate specificity of the Pnn knock down, a rescue vector was designed by incorporating three conservative nucleotide substitutions within the Pnn-shRNAi targeting sequences of the full length Pnn-GFP construct, thus generating a Pnn construct to produce mRNA that Pnn shRNAi could not target (Pnn-CS3-GFP). RESULTS: HCE-T cells were cotransfected with Pnn shRNAi and GFP vectors and after 24 and 48 h exhibited significantly reduced immunostaining for Pnn. Western blot analyses of Pnn and E-cadherin protein expression in cells transfected with Pnn-shRNAi and GFP vectors revealed marked reduction in levels of both proteins compared to those observed in cells transfected with GFP alone. The cells receiving Pnn-shRNAi appeared to be less adherent to neighboring nontransfected cells, often exhibited altered cell shape, downregulated cell adhesion and cell junction molecules, and escaped from the epithelium. The Pnn shRNAi transfected cells exhibited fewer keratin filaments anchored to desmosomes and a concurrent increase in the perinuclear bundling of filaments. SR proteins and SRm300 showed an altered distribution in the Pnn knock down cells. Cotransfection of Pnn-CS3-GFP with Pnn shRNAi demonstrated that the conservatively mutated Pnn could maintain cell-cell adhesion. CONCLUSIONS: Our results indicate that knocking down Pnn expression leads to a loss of epithelial cell-cell adhesion, changes in cell shape, and movement of Pnn shRNAi transfected cells out of the epithelium. We suggest that Pnn plays an integral role in the establishment and maintenance of epithelial cell-cell adhesion via its activity within nuclear multi-protein complexes.

Pinin/DRS/memA interacts with SRp75, SRm300 and SRrp130 in corneal epithelial cells.

PURPOSE: Pinin (Pnn/DRS/memA) is a cell-adhesion-related and nuclear protein that has been identified as central in the establishment and maintenance of corneal epithelial cell-cell adhesion. To begin the elucidation of the role of Pnn within the nucleus of corneal epithelial cells, this study was undertaken to identify the proteins that bind to Pnn. METHODS: Yeast two-hybrid analyses were performed. A human cDNA library in the pGAD-10 vector and C-terminal region of human Pnn (465-717) in a pAS2-1 vector were cotransformed into the PJ69-4A yeast strain, containing the lacZ, HIS3, and ADE2 reporter genes. To dissect domains of Pnn responsible for mediating the interaction with the identified proteins, PNN fragments were ligated with the DNA-binding domain of the pAS2-1 vector. Human corneal epithelial cells (HCE-T, RCB1384) and HEK-293 cells were cotransfected with mammalian expression vectors containing Pnn with identified interacting partners and subsequently immunostained and immunoblotted to determine expressed and endogenous proteins. RESULTS: Pnn colocalized and copurified with serine-arginine (SR) proteins. Three SR-rich proteins were identified that interact with the C-terminus of Pnn: SRp75 and SRm300, known components of spliceosome machinery, and a novel 130-kDa nuclear protein, SRrp130. All of these proteins colocalized and coimmunoprecipitated with one another and exhibited speckled nuclear distribution that aligned with components of the pre-mRNA splicing machinery. The cDNA for SRrp130 encoded a protein of 805 amino acid residues and contained multiple arginine-serine (RS) repeats but had no RNA recognition motif. Analysis of the Pnn motifs using two-hybrid system assays demonstrated that the polyserine/RS motif within Pnn plays a central but not exclusive role in mediating molecular interactions with identified SR-rich proteins. CONCLUSIONS: The results suggest that Pnn and SR-rich proteins may be part of a multiprotein complex within the nucleus and may be involved in pre-mRNA processing.