Fluid shear stress induces beta-catenin signaling in osteoblasts.

beta-Catenin plays a dual role in cells: one at cell-cell junctions and one regulating gene transcription together with TCF (T-cell Factor) in the nucleus. Recently, a role for beta-catenin in osteoblast differentiation and gene expression has begun to be elucidated. Herein we investigated the effects of fluid shear stress (FSS) on beta-catenin signaling. FSS is a well-characterized anabolic stimulus for osteoblasts; however, the molecular mechanisms for the effects of this stimulation remain largely unknown. We found that 1 hour of laminar FSS (10 dynes/cm(2)) induced translocation of beta-catenin to the nucleus and activated a TCF-reporter gene. Analysis of upstream signals that may regulate beta-catenin signaling activity revealed two potential mechanisms for increased beta-catenin signaling. First, FSS induced a transient, but significant, increase in the phosphorylation of both glycogen synthase kinase 3beta (GSK-3beta) and Akt. Second, FSS reduced the levels of beta-catenin associated with N-cadherin, suggesting that less sequestration of beta-catenin by cadherins occurs in osteoblasts subjected to FSS. Functional analysts of potential genes regulated by beta-catenin signaling in osteoblasts revealed two novel observations. First, endogenous, nuclear beta-catenin purified from osteoblasts formed a complex with a TCF -binding element in the cyclooxygenase-2 promoter, and, second, overexpression of either a constitutively active beta-catenin molecule or inhibition of GSK-3beta activity increased basal cyclooxygenase-2 levels. Together, these data demonstrate for the first time that FSS modulates the activity of both GSK-3beta and beta-catenin and that these signaling molecules regulate cyclooxygenase-2 expression in osteoblasts.

Regulation of PGE(2) and PGI(2) release from human umbilical vein endothelial cells by actin cytoskeleton.

Disruption of microfilaments in human umbilical vein endothelial cells (HUVEC) with cytochalasin D (cytD) or latrunculin A (latA) resulted in a 3.3- to 5.7-fold increase in total synthesis of prostaglandin E(2) (PGE(2)) and a 3.4- to 6.5-fold increase in prostacyclin (PGI(2)) compared with control cells. Disruption of the microtubule network with nocodazole or colchicine increased synthesis of PGE(2) 1.7- to 1.9-fold and PGI(2) 1.9- to 2.0-fold compared with control cells. Interestingly, however, increased release of PGE(2) and PGI(2) from HUVEC into the media occurred only when microfilaments were disrupted. CytD treatment resulted in 6.7-fold more PGE(2) and 3.8-fold more PGI(2) released from HUVEC compared with control cells; latA treatment resulted in 17.7-fold more PGE(2) and 11.2-fold more PGI(2) released compared with control cells. Both increased synthesis and release of prostaglandins in response to all drug treatments were completely inhibited by NS-398, a specific inhibitor of cyclooxygenase-2 (COX-2). Disruption of either microfilaments using cytD or latA or of microtubules using nocodazole or colchicine resulted in a significant increase in COX-2 protein levels, suggesting that the increased synthesis of prostaglandins in response to drug treatments may result from increased activity of COX-2. These results, together with studies demonstrating a vasoprotective role for prostaglandins, suggest that the cytoskeleton plays an important role in maintenance of endothelial barrier function by regulating prostaglandin synthesis and release from HUVEC.

Assembly of desmosomal cadherins into desmosomes is isoform dependent.

Desmosomes are intercellular adhesive junctions that exhibit cell- and differentiation-specific differences in their molecular composition. In complex epithelia, desmosomes contain multiple representatives of the desmosomal cadherin family, which includes three desmogleins and three desmocollins. Rules governing the assembly of desmosomal cadherin isoforms into desmosomes of different cell types are unknown. Here we compared the assembly properties of desmoglein 2 (Dsg2) and desmocollin 2 (Dsc2), which are widely expressed, with Dsg1 and Dsc1, which are expressed in the differentiated layers of complex epithelia, by introducing myc-tagged forms into simple and squamous epithelial cells that do not express Dsg1 or Dsc1. Dsc2.myc and Dsg2.myc assembled efficiently into desmosomes in every cell type in spite of significant shifts in the stoichiometric relationship between desmogleins and desmocollins. In contrast, Dsc1a.myc, Dsc1b.myc, and Dsg1.myc did not stably incorporate into desmosomes in any line. Coexpression of Dsc1a.myc or Dsc1b.myc and Dsg1.myc did not lead to their colocalization and failed to enhance incorporation of either cadherin into desmosomes. Dsg1.myc, but not Dsc1a, Dsc1b, disrupted desmosome assembly in a cell-type-specific manner, and disruption correlated with the recruitment of Dsg1.myc, but not Dsc1a or Dsc1b, into a Triton-insoluble pool. The plakoglobin:E-cadherin ratio decreased in Dsg1-expressing cells with disrupted desmosomes, but a decrease was also observed in a Dsc1a line. Thus, a modest reduction of plakoglobin associated with E-cadherin is apparently not sufficient to disrupt desmosome assembly. Our results demonstrate that desmosome assembly tolerates large shifts in cadherin stoichiometry, but is sensitive to isoform-specific differences exhibited by desmogleins and desmocollins.

Desmosomes: intercellular adhesive junctions specialized for attachment of intermediate filaments.

Cell-cell adhesion is thought to play important roles in development, in tissue morphogenesis, and in the regulation of cell migration and proliferation. Desmosomes are adhesive intercellular junctions that anchor the intermediate filament network to the plasma membrane. By functioning both as an adhesive complex and as a cell-surface attachment site for intermediate filaments, desmosomes integrate the intermediate filament cytoskeleton between cells and play an important role in maintaining tissue integrity. Recent observations indicate that tissue integrity is severely compromised in autoimmune and genetic diseases in which the function of desmosomal molecules is impaired. In addition, the structure and function of many of the desmosomal molecules have been determined, and a number of the molecular interactions between desmosomal proteins have now been elucidated. Finally, the molecular constituents of desmosomes and other adhesive complexes are now known to function not only in cell adhesion, but also in the transduction of intracellular signals that regulate cell behavior.

Contributions of extracellular and intracellular domains of full length and chimeric cadherin molecules to junction assembly in epithelial cells.

The integrity of cell-cell junctions in epithelial cells depends on functional interactions of both extracellular and intracellular domains of cadherins with other junction proteins. To examine the roles of the different domains of E-cadherin and desmoglein in epithelial junctions, we stably expressed full length desmoglein 1 and chimeras of E-cadherin and desmoglein 1 in A431 epithelial cells. Full length desmoglein 1 was able to incorporate into or disrupt endogenous desmosomes depending on expression level. Each of the chimeric cadherin molecules exhibited distinct localization patterns at the cell surface. A chimera of the desmoglein 1 extracellular domain and the E-cadherin intracellular domain was distributed diffusely at the cell surface while the reverse chimera, comprising the E-cadherin extracellular domain and the desmoglein 1 intracellular domain, localized in large, sometimes contiguous patches at cell-cell interfaces. Nevertheless, both constructs disrupted desmosome assembly. Expression of constructs containing the desmoglein 1 cytoplasmic domain resulted in approximately a 3-fold decrease in E-cadherin bound to plakoglobin and a 5- to 10-fold reduction in the steady-state levels of the endogenous desmosomal cadherins, desmoglein 2 and desmocollin 2, possibly contributing to the dominant negative effect of the desmoglein 1 tail. In addition, biochemical analysis of protein complexes in the stable lines revealed novel in vivo protein interactions. Complexes containing beta-catenin and desmoglein 1 were identified in cells expressing constructs containing the desmoglein 1 tail. Furthermore, interactions were identified between endogenous E-cadherin and the chimera containing the E-cadherin extracellular domain and the desmoglein 1 intracellular domain providing in vivo evidence for previously predicted lateral interactions of E-cadherin extracellular domains.

The expression of desmoglein isoforms in cultured human keratinocytes is regulated by calcium, serum, and protein kinase C.

Three desmoglein (Dsg) isoforms are expressed in a differentiation-specific fashion in the epidermis, with Dsg2 being basal, Dsg3 (pemphigus vulgaris antigen) basal and spinous, and Dsg1 (pemphigus foliaceus antigen) predominantly granular. To better understand the mechanism(s) regulating Dsg isoform expression, we examined the expression pattern of Dsg1, Dsg2, and Dsg3 in normal human epidermal keratinocytes (NHEKs), the immortalized, nontumorigenic HaCaT cell line, and several squamous cell carcinoma cell lines (SCC-9, SCC-12F, SCC-13, and SCC-25). In all cells, the accumulation of high Dsg protein levels required calcium and was not observed in low calcium (0.05-0.07 mM) media. NHEKs expressed Dsg1 in all media tested, consistent with their normal differentiation capacity. HaCaT and SCC-25 also expressed Dsg1; however, the presence of serum in the media dramatically decreased Dsg1 protein levels. Serum also inhibited Dsg1 mRNA levels in HaCaT cells. Dsg1 was not detected in extracts from SCC-9, SCC-12F, and SCC-13 under any conditions. Since activation of protein kinase C (PKC) is involved in keratinocyte differentiation, we evaluated the effects of PKC down-regulation on Dsg isoform expression. Long-term treatment with either the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) or bryostatin 1 inhibited levels of Dsg1 and Dsg3, but not Dsg2 in NHEKs and HaCaT cells. Chronic TPA also decreased Dsg1 and Dsg3 mRNA levels in NHEKs, further supporting a role for PKC activation in the expression of the suprabasal Dsg1 and Dsg3. These results identify several regulatory mechanisms by which the differentiation-specific pattern of desmosomal cadherins is established in the epidermis.