Spatial expression of the genome: the signal hypothesis at forty.

The signal hypothesis, formulated by Günter Blobel and David Sabatini in 1971, and elaborated by Blobel and his colleagues between 1975 and 1980, fundamentally expanded our view of cells by introducing the concept of topogenic signals. Cells were no longer just morphological entities with compartmentalized biochemical functions; they were now active participants in the creation and perpetuation of their own form and identity, the decoders of linear genetic information into three dimensions.

BAMBI is a novel HIF1-dependent modulator of TGFß-mediated disruption of cell polarity during hypoxia.

Hypoxia and loss of cell polarity are common features of malignant carcinomas. Hypoxia-inducible factor 1 (HIF1) is the major regulator of cellular hypoxia response and mediates the activation of ∼300 genes. Increased HIF1 signaling is known to be associated with epithelial-mesenchymal transformation. Here, we report that hypoxia disrupts polarized epithelial morphogenesis of MDCK cells in a HIF1α-dependent manner by modulating the transforming growth factor-ß (TGFß) signaling pathway. Analysis of potential HIF1 targets in the TGFß pathway identified the bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI), a transmembrane glycoprotein related to the type I receptors of the TGFß family, whose expression was essentially lost in HIF1-depleted cells. Similar to what was observed in HIF1-deficient cells, BAMBI-depleted cells failed to efficiently activate TGFß signaling and retained epithelial polarity during hypoxia. Taken together, we show that hypoxic conditions promote TGFß signaling in a HIF1-dependent manner and BAMBI is identified in this pathway as a novel HIF1-regulated gene that contributes to hypoxia-induced loss of epithelial polarity.

The Heuristic of Form: Mitochondrial Morphology and the Explanation of Oxidative Phosphorylation.

In the 1950s and 1960s, the search for the mechanism of oxidative phosphorylation by biochemists paralleled the description of mitochondrial form by George Palade and Fritiof Sjöstrand using electron microscopy. This paper explores the extent to which biochemists studying oxidative phosphorylation took mitochondrial form into account in the formulation of hypotheses, design of experiments, and interpretation of results. By examining experimental approaches employed by the biochemists studying oxidative phosphorylation, and their interactions with Palade, I suggest that use of mitochondrial form as a guide to experimentation and interpretation varied considerably among investigators. Most notably, Peter Mitchell, whose chemiosmotic hypothesis was ultimately the basis of the correct mechanism of oxidative phosphorylation, incorporated crucial aspects of mitochondrial form into his model that others failed to recognize. I discuss these historical observations in terms of the background and training of the biochemists, as well as a proposed heuristic of form, whose use may increase the possibility that biologically meaningful molecular mechanisms will be discovered.

Activated PKC{delta} and PKC{epsilon} inhibit epithelial chloride secretion response to cAMP via inducing internalization of the Na+-K+-2Cl- cotransporter NKCC1.

The basolateral Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) is a key determinant of transepithelial chloride secretion and dysregulation of chloride secretion is a common feature of many diseases including secretory diarrhea. We have previously shown that activation of protein kinase C (PKC) markedly reduces transepithelial chloride secretion in human colonic T84 cells, which correlates with both functional inhibition and loss of the NKCC1 surface expression. In the present study, we defined the specific roles of PKC isoforms in regulating epithelial NKCC1 and chloride secretion utilizing adenoviral vectors that express shRNAs targeting human PKC isoforms (α, δ, ε) (shPKCs) or LacZ (shLacZ, non-targeting control). After 72 h of adenoviral transduction, protein levels of the PKC isoforms in shPKCs-T84 cells were decreased by ∼90% compared with the shLacZ-control. Activation of PKCs by phorbol 12-myristate 13-acetate (PMA) caused a redistribution of NKCC1 immunostaining from the basolateral membrane to intracellular vesicles in both shLacZ- and shPKCα-T84 cells, whereas the effect of PMA was not observed in shPKCδ- and shPKCε- cells. These results were further confirmed by basolateral surface biotinylation. Furthermore, activation of PKCs by PMA inhibited cAMP-stimulated chloride secretion in the uninfected, shLacZ- and shPKCα-T84 monolayers, but the inhibitory effect was significantly attenuated in shPKCδ- and shPKCε-T84 monolayers. In conclusion, the activated novel isoforms PKCδ or PKCε, but not the conventional isoform PKCα, inhibits transepithelial chloride secretion through inducing internalization of the basolateral surface NKCC1. Our study reveals that the novel PKC isoform-regulated NKCC1 surface expression plays an important role in the regulation of chloride secretion.

Phorbol 12-myristate 13-acetate-induced endocytosis of the Na-K-2Cl cotransporter in MDCK cells is associated with a clathrin-dependent pathway.

In secretory epithelial cells, the basolateral Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) plays a major role in salt and fluid secretion. Our laboratory has identified NKCC1 surface expression as an important regulatory mechanism for Cl(-) secretion in the colonic crypt cell line T84, a process also present in native human colonic crypts. We previously showed that activation of protein kinase C (PKC) by carbachol and phorbol 12-myristate 13-acetate (PMA) decreases NKCC1 surface expression in T84 cells. However, the specific endocytic entry pathway has not been defined. We used a Madin-Darby canine kidney (MDCK) cell line stably transfected with enhanced green fluorescent protein (EGFP)-NKCC1 to map NKCC1 entry during PMA exposure. At given times, we fixed and stained the cells with specific markers (e.g., dynamin II, clathrin heavy chain, and caveolin-1). We also used chlorpromazine, methyl-beta-cyclodextrin, amiloride, and dynasore, blockers of the clathrin, caveolin, and macropinocytosis pathways and the vesicle "pinchase" dynamin, respectively. We found that PMA caused dose- and time-dependent NKCC1 endocytosis. After 2.5 min of PMA exposure, approximately 80% of EGFP-NKCC1 endocytic vesicles colocalized with clathrin and approximately 40% colocalized with dynamin II and with the transferrin receptor, the uptake of which is also mediated by clathrin-coated vesicles. We did not observe significant colocalization of EGFP-NKCC1 endocytic vesicles with caveolin-1, a marker of the caveolae-mediated endocytic pathway. We quantified the effect of each inhibitor on PMA-induced EGFP-NKCC1 endocytosis and found that only chlorpromazine and dynasore caused significant inhibition compared with the untreated control (61% and 25%, respectively, at 2.5 min). Together, these results strongly support the conclusion that PMA-stimulated NKCC1 endocytosis is associated with a clathrin pathway.

Regulated synthesis and functions of laminin 5 in polarized madin-darby canine kidney epithelial cells.

Renal tubular epithelial cells synthesize laminin (LN)5 during regeneration of the epithelium after ischemic injury. LN5 is a truncated laminin isoform of particular importance in the epidermis, but it is also constitutively expressed in a number of other epithelia. To investigate the role of LN5 in morphogenesis of a simple renal epithelium, we examined the synthesis and function of LN5 in the spreading, proliferation, wound-edge migration, and apical-basal polarization of Madin-Darby canine kidney (MDCK) cells. MDCK cells synthesize LN5 only when subconfluent, and they degrade the existing LN5 matrix when confluent. Through the use of small-interfering RNA to knockdown the LN5 alpha3 subunit, we were able to demonstrate that LN5 is necessary for cell proliferation and efficient wound-edge migration, but not apical-basal polarization. Surprisingly, suppression of LN5 production caused cells to spread much more extensively than normal on uncoated surfaces, and exogenous keratinocyte LN5 was unable to rescue this phenotype. MDCK cells also synthesized laminin alpha5, a component of LN10, that independent studies suggest may form an assembled basal lamina important for polarization. Overall, our findings indicate that LN5 is likely to play an important role in regulating cell spreading, migration, and proliferation during reconstitution of a continuous epithelium.

The protein kinase A pathway contributes to Hg2+-induced alterations in phosphorylation and subcellular distribution of occludin associated with increased tight junction permeability of salivary epithelial cell monolayers.

Hg(2+) is commonly used as an inhibitor of many aquaporins during measurements of transcellular water transport. To investigate whether it could also act on the paracellular water transport pathway, we asked whether addition of Hg(2+) affected transport of radiolabeled probes through tight junctions of a salivary epithelial cell monolayer. Inclusion of 1 mM Hg(2+) decreased transepithelial electrical resistance by 8-fold and augmented mannitol and raffinose flux by 13-fold, which translated into an estimated 44% increase in pore radius at the tight junction. These Hg(2+)-induced effects could be partially blocked by the protein kinase A (PKA) inhibitor N-[2-((p-bromocinnamyl) amino) ethyl]-5-isoquinolinesulfonamide, 2HCl (H89), suggesting that both-PKA dependent and PKA-independent mechanisms contribute to tight junction regulation. Western blot analyses showed a 2-fold decrease in tight junction-associated occludin after Hg(2+) treatment and the presence of a novel hyperphosphorylated form of occludin in the cytoplasmic fraction. These findings were corroborated by confocal imaging. The results from this study reveal a novel contribution of the PKA pathway in Hg(2+)-induced regulation of tight junction permeability in the salivary epithelial barrier. Therapeutically, this could be explored for pharmacological intervention in the treatment of dry mouth, Sjögren's syndrome, and possibly other disorders of fluid transport.

Beta1-integrin orients epithelial polarity via Rac1 and laminin.

Epithelial cells polarize and orient polarity in response to cell-cell and cell-matrix adhesion. Although there has been much recent progress in understanding the general polarizing machinery of epithelia, it is largely unclear how this machinery is controlled by the extracellular environment. To explore the signals from cell-matrix interactions that control orientation of cell polarity, we have used three-dimensional culture systems in which Madin-Darby canine kidney (MDCK) cells form polarized, lumen-containing structures. We show that interaction of collagen I with apical beta1-integrins after collagen overlay of a polarized MDCK monolayer induces activation of Rac1, which is required for collagen overlay-induced tubulocyst formation. Cysts, comprised of a monolayer enclosing a central lumen, form after embedding single cells in collagen. In those cultures, addition of a beta1-integrin function-blocking antibody to the collagen matrix gives rise to cysts that have defects in the organization of laminin into the basement membrane and have inverted polarity. Normal polarity is restored by either expression of activated Rac1, or the inclusion of excess laminin-1 (LN-1). Together, our results suggest a signaling pathway in which the activation of beta1-integrins orients the apical pole of polarized cysts via a mechanism that requires Rac1 activation and laminin organization into the basement membrane.

Mechanical stimulation increases collagen type I and collagen type III gene expression of stem cell-collagen sponge constructs for patellar tendon repair.

Our group has shown that mechanical stimulation increases the stiffness of stem cell-collagen sponge constructs at 14 days in culture and subsequent rabbit patellar tendon repairs at 12 weeks postsurgery. What remains unclear is which genes might be responsible for this increase in stiffness. Therefore, the objective of this study was to determine how a tensile stimulus affects the gene expression of stem cell-collagen sponge constructs used to repair rabbit central patellar tendon defects. Tissue-engineered constructs were created by seeding mesenchymal stem cells (MSCs) from 10 adult rabbits at 0.14 x 10(6) cells/construct in type I collagen sponges. Half of the constructs were mechanically stimulated once every 5 min for 8 h/d to a peak strain of 2.4% for 2 weeks. The other half remained in an incubator without mechanical stimulation for 2 weeks. After 14 days in culture, half of the stimulated and nonstimulated constructs were prepared to determine the expression of collagen type I, collagen type III, decorin, fibronectin, and glyceraldehyde-3-phosphate dehydrogenase genes using real-time quantitative reverse transcriptase polymerase chain reaction. The remaining constructs were mechanically tested to determine their mechanical properties. Two weeks of in vitro mechanical stimulation significantly increased collagen type I and collagen type III gene expression of the stem cell-collagen sponge constructs. Stimulated constructs showed 3 and 4 times greater collagen type I (p = 0.0001) and collagen type III gene expression (p = 0.001) than nonstimulated controls. Stimulated constructs also had 2.5 times the linear stiffness and 4 times the linear modulus of nonstimulated constructs. However, mechanical stimulation did not significantly increase decorin or fibronectin gene expression (p = 0.2) after 14 days in culture. This study shows that mechanical stimulation of cell-sponge constructs produces similar increases in the expression of 2 structural genes, as well as linear stiffness and linear modulus.

The secretory pathway at 50: a golden anniversary for some momentous grains of silver.

The secretory pathway along which newly synthesized secretory and membrane proteins traffic through the cell was revealed in two articles published 50 years ago. This discovery was the culmination of decades of effort to unite the power of biochemical and morphological methodologies in order to elucidate the dynamic nature of the cell's biosynthetic machinery. The secretory pathway remains a central paradigm of modern cell biology. Its elucidation 50 years ago inspired tremendous multidisciplinary and on-going efforts to understand the machinery that makes it run, the adaptations that permit it to serve the needs of specialized cell types, and the pathological consequences that arise when it is perturbed.

Laminins in Epithelial Cell Polarization: Old Questions in Search of New Answers.

Laminin, a basement membrane protein discovered in 1979, was shortly thereafter implicated in the polarization of epithelial cells in both mammals and a variety of lower organisms. To transduce a spatial cue to the intrinsic polarization machinery, laminin must polymerize into a dense network that forms the foundation of the basement membrane. Evidence suggests that activation of the small GTPase Rac1 by ß1-integrins mobilizes laminin-binding integrins and dystroglycan to consolidate formation of the laminin network and initiate rearrangements of both the actin and microtubule cytoskeleton to help establish the apicobasal axis. A key coordinator of spatial signals from laminin is the serine-threonine kinase Par-1, which is known to affect dystroglycan availability, microtubule and actin organization, and lumen formation. The signaling protein integrin-linked kinase (ILK) may also play a role. Despite significant advances, knowledge of the mechanism by which assembled laminin produces a spatial signal remains fragmentary, and much more research into the complex functions of laminin in polarization and other cellular processes is needed.

αV-integrins are required for mechanotransduction in MDCK epithelial cells.

The properties of epithelial cells within tissues are regulated by their immediate microenvironment, which consists of neighboring cells and the extracellular matrix (ECM). Integrin heterodimers orchestrate dynamic assembly and disassembly of cell-ECM connections and thereby convey biochemical and mechanical information from the ECM into cells. However, the specific contributions and functional hierarchy between different integrin heterodimers in the regulation of focal adhesion dynamics in epithelial cells are incompletely understood. Here, we have studied the functions of RGD-binding αV-integrins in a Madin Darby Canine Kidney (MDCK) cell model and found that αV-integrins regulate the maturation of focal adhesions (FAs) and cell spreading. αV-integrin-deficient MDCK cells bound collagen I (Col I) substrate via α2ß1-integrins but failed to efficiently recruit FA components such as talin, focal adhesion kinase (FAK), vinculin and integrin-linked kinase (ILK). The apparent inability to mature α2ß1-integrin-mediated FAs and link them to cellular actin cytoskeleton led to disrupted mechanotransduction in αV-integrin deficient cells seeded onto Col I substrate.

Laminin 511 partners with laminin 332 to mediate directional migration of Madin-Darby canine kidney epithelial cells.

Sustained directional migration of epithelial cells is essential for regeneration of injured epithelia. Front-rear polarity of migrating cells is determined by local activation of a signaling network involving Cdc42 and other factors in response to spatial cues from the environment, the nature of which are obscure. We examined the roles of laminin (LM)-511 and LM-332, two structurally different laminin isoforms, in the migration of Madin-Darby canine kidney cells by suppressing expression of their α subunits using RNA interference. We determined that knockdown of LM-511 inhibits directional migration and destabilizes cell-cell contacts, in part by disturbing the localization and activity of the polarization machinery. Suppression of integrin α3, a laminin receptor subunit, in cells synthesizing normal amounts of both laminins has a similar effect as knockdown of LM-511. Surprisingly, simultaneous suppression of both laminin α5 and laminin α3 restores directional migration and cell-cell contact stability, suggesting that cells recognize a haptotactic gradient formed by a combination of laminins.

Autocrine transforming growth factor-{beta}1 activation mediated by integrin {alpha}V{beta}3 regulates transcriptional expression of laminin-332 in Madin-Darby canine kidney epithelial cells.

Laminin (LM)-332 is an extracellular matrix protein that plays a structural role in normal tissues and is also important in facilitating recovery of epithelia from injury. We have shown that expression of LM-332 is up-regulated during renal epithelial regeneration after ischemic injury, but the molecular signals that control expression are unknown. Here, we demonstrate that in Madin-Darby canine kidney (MDCK) epithelial cells LM-332 expression occurs only in subconfluent cultures and is turned-off after a polarized epithelium has formed. Addition of active transforming growth factor (TGF)-ß1 to confluent MDCK monolayers is sufficient to induce transcription of the LM α3 gene and LM-332 protein expression via the TGF-ß type I receptor (TßR-I) and the Smad2-Smad4 complex. Significantly, we show that expression of LM-332 in MDCK cells is an autocrine response to endogenous TGF-ß1 secretion and activation mediated by integrin αVß3 because neutralizing antibodies block LM-332 production in subconfluent cells. In confluent cells, latent TGF-ß1 is secreted apically, whereas TßR-I and integrin αVß3 are localized basolaterally. Disruption of the epithelial barrier by mechanical injury activates TGF-ß1, leading to LM-332 expression. Together, our data suggest a novel mechanism for triggering the production of LM-332 after epithelial injury.

Dynamic regulation of Na(+)-K(+)-2Cl(-) cotransporter surface expression by PKC-{epsilon} in Cl(-)--secretory epithelia.

In secretory epithelia, activation of PKC by phorbol ester and carbachol negatively regulates Cl(-) secretion, the transport event of secretory diarrhea. Previous studies have implicated the basolateral Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) as a target of PKC-dependent inhibition of Cl(-) secretion. In the present study, we examined the regulation of surface expression of NKCC1 in response to the activation of PKC. Treatment of confluent T84 intestinal epithelial cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (PMA) reduced the amount of NKCC1 accessible to basolateral surface biotinylation. Loss of cell surface NKCC1 was due to internalization as shown by 1) the resistance of biotinylated NKCC1 to surface biotin stripping after incubation with PMA and 2) indirect immunofluorescent labeling. PMA-induced internalization of NKCC1 is dependent on the epsilon-isoform of PKC as determined on the basis of sensitivity to a panel of PKC inhibitors. The effect of PMA on surface expression of NKCC1 was specific because PMA did not significantly alter the amount of Na(+)-K(+)-ATPase or E-cadherin available for surface biotinylation. After extended PMA exposure (>2 h), NKCC1 became degraded in a proteasome-dependent fashion. Like PMA, carbachol reduced the amount of NKCC1 accessible to basolateral surface biotinylation in a PKC-epsilon-dependent manner. However, long-term exposure to carbachol did not result in degradation of NKCC1; rather, NKCC1 that was internalized after exposure to carbachol was recycled back to the cell membrane. PKC-epsilon-dependent alteration of NKCC1 surface expression represents a novel mechanism for regulating Cl(-) secretion.

Differential subcellular targeting of PKC-epsilon in response to pharmacological or ischemic stimuli in intestinal epithelia.

Ischemia is the central pathogenic factor underlying a spectrum of intestinal disorders. The study of the cellular signaling responses to ischemic stress in nonepithelial cells has progressed substantially in the previous several years, but little is known about the response in epithelial cells. Unique features of the epithelial response to ischemic stress suggest differential regulation with regards to signaling. The PKC family of proteins has been implicated in ischemic stress in nonepithelial systems. The role of PKC isoforms in chemical ischemia in intestinal epithelial cells is evaluated in this study. Additionally, the phosphorylation of the F-actin cross-linking protein myristoylated alanine-rich C kinase substrate (MARCKS) is also studied. Chemical ischemia resulted in the transient activation of only the isoform PKC-epsilon as detected by translocation employing the subcellular fractionation technique. The pharmacological agonists phorbol 12-myristate 13-acetate and carbachol also led to the translocation of PKC-epsilon. By immunofluoresence, MARCKS is noted to be located at the lateral membrane under control conditions. In response to carbachol, MARCKS translocates to the cytosol, indicating its phosphorylation, which is additionally confirmed biochemically. Consistent with this observation, carbachol induces the translocation of PKC-epsilon to proximity with MARCKS at the lateral membrane. In response to chemical ischemia, MARCKS fails to translocate and phosphorylation does not increase. Additionally, the translocation of PKC-epsilon is not to the lateral membrane but rather basally. The data suggest that the differential translocation of PKC-epsilon in response to pharmacological agonists versus ischemic stress may lead to different effects on downstream targets.

Induction of a laminin isoform and alpha(3)beta(1)-integrin in renal ischemic injury and repair in vivo.

Ischemic injury to the kidney, a major cause of acute renal failure, leads to the detachment and loss of numerous tubular epithelial cells. Integrin-laminin interactions may promote regeneration of the damaged epithelium by influencing kidney epithelial cell adhesion and differentiation. Laminins are major structural components of basement membranes. Of the various laminin isoforms, laminin-5 is of particular interest because of its proposed role in the healing of skin wounds. In this study, we investigate the expression of laminin-5 in rat kidney after unilateral ischemia. Using a polyclonal antibody generated against laminin-5, we find that immunostaining is confined to the basement membranes of collecting ducts in the papilla and the major and minor calyces in normal kidney. With injury and regeneration, however, immunostaining becomes much more intense and widespread in basement membranes along the nephron. Immunoblotting of ischemic kidney extracts reveals significantly increased expression of a polypeptide of approximately 220 kDa, possibly corresponding to a precursor of one of the three laminin-5 chains. Immunoblotting and immunostaining also demonstrate significantly increased expression and altered localization of the alpha(3)-integrin subunit, a receptor for laminin-5. These results indicate that there is induction of a laminin isoform, possibly laminin-5, and alpha(3)beta(1)-integrin in the ischemic kidney and may implicate this receptor-ligand combination in the pathogenesis of acute renal failure and/or repair of the injured kidney epithelium.

Integrins in epithelial cell polarity: using antibodies to analyze adhesive function and morphogenesis.

Epithelial cells polarize in response to cell-substratum and cell-cell adhesive interactions. Contacts between cells and proteins of the extracellular matrix are mediated by integrin receptors. Of the 24 recognized integrin heterodimers, epithelial cells typically express four or more distinct integrins, with the exact complement dependent on the tissue of origin. Investigation of the roles of integrins in epithelial cell polarization has depended on the use of function-blocking antibodies both to determine ligand specificity of individual integrins and to disrupt and redirect normal morphogenesis. In this article we describe techniques for employing function-blocking anti-integrin antibodies in adhesion assays of the polarized Madin-Darby canine kidney (MDCK) cell line and to demonstrate the involvement of beta1 integrins in collagen-induced tubulocyst formation. These techniques can be easily expanded to other antibodies and epithelial cell lines to characterize specific functions of individual integrins in epithelial morphogenesis.

Regulation of MDCK cell-substratum adhesion by RhoA and myosin light chain kinase after ATP depletion.

The attachment of epithelial cells to the extracellular matrix substratum is essential for their differentiation and polarization. Despite this, the precise adhesion mechanism and its regulation are poorly understood. In the kidney, an ischemic insult causes renal tubular epithelial cells to detach from the basement membrane, even though they remain viable. To understand this phenomenon, and to probe the regulation of epithelial cell attachment, we used a model system consisting of newly adherent Madin-Darby canine kidney (MDCK) cells subjected to ATP depletion to mimic ischemic injury. We found that MDCK cells detach from collagen I after 60 min of ATP depletion but reattach when resupplied with glucose. Detachment is not caused by degradation or endocytosis of beta(1)-integrins, which mediate attachment to collagen I. Basal actin filaments and paxillin-containing adhesion complexes are disrupted by ATP depletion and quickly reform on glucose repletion. However, partial preservation of basal actin by overexpression of constitutively active RhoA does not significantly affect cell detachment. Furthermore, Y-27632, an inhibitor of the RhoA effector Rho-kinase, does not prevent reattachment of cells on glucose addition, even though reformation of central stress fibers and large adhesion complexes is blocked. In contrast, reattachment of ATP-depleted cells and detachment of cells not previously subjected to ATP depletion are prevented by ML-7, an inhibitor of myosin light chain kinase (MLCK). We conclude that initial adherence of MDCK cells to a collagen I substratum is mediated by peripheral actin filaments and adhesion complexes regulated by MLCK but not by stress fibers and adhesion complexes controlled by RhoA.