Cadherins and cancer: how does cadherin dysfunction promote tumor progression?

It has long been recognized that the cell-cell adhesion receptor, E-cadherin, is an important determinant of tumor progression, serving as a suppressor of invasion and metastasis in many contexts. Yet how the loss of E-cadherin function promotes tumor progression is poorly understood. In this review, we focus on three potential underlying mechanisms: the capacity of E-cadherin to regulate beta-catenin signaling in the canonical Wnt pathway; its potential to inhibit mitogenic signaling through growth factor receptors and the possible links between cadherins and the molecular determinants of epithelial polarity. Each of these potential mechanisms provides insights into the complexity that is likely responsible for the tumor-suppressive action of E-cadherin.

Evidence-based systematic review on the efficacy and safety of botulinum toxin-A therapy in post-stroke spasticity.

BACKGROUND: Botulinum toxin type A (BoNTA) has been suggested as an effective anti-spastic drug. In this article, we summarized the data of randomized, placebo-controlled, double- blind trials and conducted a meta-analysis to assess if BoNTA is an adequate treatment for spasticity following stroke. OBJECTIVES: To evaluate the relevant literature and assess the effectiveness and safety of BoNTA in (1) reducing spasticity based on mean change in the Modified Ashworth Scale (MAS) for upper and lower limb spasticity from baseline; (2) reducing spasticity based on the percent of patients having > or = 1point(s) change in the MAS; (3) improving the patient's or caregivers' Global Assessment Scale (GAS); and (4) total adverse events. METHOD: We selected all randomized, placebo controlled, double-blind clinical trials or previous meta-analyses evaluating the efficacy and safety of BoNTA (Botox or Dysport) for the treatment of spasticity in both upper and lower limbs after stroke. Validity assessment of studies was performed, and Revman 4.2.7 from Cochrane Collaboration and SPSS (statistical package for the social sciences), v 9.0, were applied for analysis. RESULTS: Overall analysis showed clinical improvement between baseline and 4-6 weeks after application of BoNTA of the patient's spasticity score using the MAS (weighted mean difference [WMD] = 0.87, 95% CI = 0.52-1.22). The odds ratio of the MAS spasticity score showing one or more points improvement at 4-6 weeks after giving BoNTA showed clinically significant improvement (OR = 4.5, 95% CI = 2.79-7.25). The odds ratio of having an improved GAS at 4-6 weeks after injecting BoNTA showed clinically significant improvement (Odds ratio = 5.85, 95% CI = 3.12-10.95). The odds ratio of having an adverse event during the entire study did not show any significant difference between placebo and BoNTA (odds ratio = 0.84, 95% CI = 0.55-1.28). REVIEWERS' CONCLUSIONS: BoNTA improves muscle tone in upper and lower limb spasticity following stroke. A global assessment of improvement was noted by the patients or the caregivers following BoNTA injection. BoNTA is considered a safe therapeutic agent.

Gab2 and Src co-operate in human mammary epithelial cells to promote growth factor independence and disruption of acinar morphogenesis.

The Gab2 docking protein is a target of several oncogenic protein tyrosine kinases and potentiates activation of the Ras/extracellular signal regulated kinase and phosphatidylinositol 3-kinase (PI3-kinase) pathways. Since Gab2 is phosphorylated by c-Src, and both proteins are overexpressed in breast cancers, we have determined the biological consequences of their co-expression in the immortalized human mammary epithelial cell line MCF-10A. While overexpression of c-Src did not affect acinar morphogenesis or growth factor dependence in three-dimensional culture, c-Src co-operated with Gab2 to promote epidermal growth factor (EGF)-independent acinar growth. In contrast, expression of v-Src or the activated mutant c-SrcY527F led to a spectrum of aberrant phenotypes ranging from spheroids with incomplete luminal clearance to highly disrupted, dispersed structures. Gab2 co-expression shifted the phenotypic distribution towards the dispersed phenotype, an effect not observed with a Gab2 mutant unable to bind the p85 subunit of PI3-kinase (Gab2Deltap85). In v-Src-expressing cells, Gab2, but not Gab2Deltap85, significantly decreased E-cadherin adhesive strength without altering its surface expression. Gab2 associated with E-cadherin in the presence and absence of v-Src, indicating that the ability of Gab2 to weaken the strength of cell-cell contacts may reflect enhanced activation of PI3-kinase at adherens junctions. Gab2 also increased migration and invasion of these cells in transwell assays, but these effects were p85-independent. Overall, these findings demonstrate a novel mechanism whereby Gab2 may promote metastatic spread and indicate that Gab2 may play several roles during breast cancer progression.

Initiation of cell locomotility is a morphogenetic checkpoint in thyroid epithelial cells regulated by ERK and PI3-kinase signals.

Epithelial locomotility is a fundamental determinant of tissue patterning that is subject to strict physiological regulation. The current study sought to identify cellular signals that initiate cell migration in cultured thyroid epithelial cells. Porcine thyroid cells cultured as 3-dimensional follicles convert to 2-dimensional monolayers when deprived of agents that stimulate cAMP/PKA signaling. This morphogenetic event is driven by the activation of cell-on-substrate locomotility, providing a convenient assay for events that regulate the initiation of locomotion. In this system, the extracellular signal regulated kinase (ERK) pathway became activated as follicles converted to monolayer, as demonstrated by immunoblotting for activation-specific phosphorylation and nuclear accumulation of ERK. Inhibition of ERK activation using the drug PD98059 effectively prevented cells from beginning to migrate. PD98059 inhibited cell spreading, actin filament reorganization and the assembly of focal adhesions, cellular events that mediate the initiation of thyroid cell locomotility. Akt (PKB) signaling was also activated during follicle-to-monolayer conversion and the phosphoinositide 3-kinase (PI3-kinase) inhibitor, wortmannin, also blocked the initiation of cell movement. Wortmannin did not, however, block activation of ERK signaling. These findings, therefore, identify the ERK and PI3-kinase signaling pathways as important stimulators of thyroid cell locomotility. These findings are incorporated into a model where the initiation of thyroid cell motility constitutes a morphogenetic checkpoint regulated by coordinated changes in stimulatory (ERK, PI3-kinase) and tonic inhibitory (cAMP/PKA) signaling pathways.

A dileucine motif targets E-cadherin to the basolateral cell surface in Madin-Darby canine kidney and LLC-PK1 epithelial cells.

E-cadherin is a major adherens junction protein of epithelial cells, with a central role in cell-cell adhesion and cell polarity. Newly synthesized E-cadherin is targeted to the basolateral cell surface. We analyzed targeting information in the cytoplasmic tail of E-cadherin by utilizing chimeras of E-cadherin fused to the ectodomain of the interleukin-2alpha (IL-2alpha) receptor expressed in Madin-Darby canine kidney and LLC-PK(1) epithelial cells. Chimeras containing the full-length or membrane-proximal half of the E-cadherin cytoplasmic tail were correctly targeted to the basolateral domain. Sequence analysis of the membrane-proximal tail region revealed the presence of a highly conserved dileucine motif, which was analyzed as a putative targeting signal by mutagenesis. Elimination of this motif resulted in the loss of Tac/E-cadherin basolateral localization, pinpointing this dileucine signal as being both necessary and sufficient for basolateral targeting of E-cadherin. Truncation mutants unable to bind beta-catenin were correctly targeted, showing, contrary to current understanding, that beta-catenin is not required for basolateral trafficking. Our results also provide evidence that dileucine-mediated targeting is maintained in LLC-PK(1) cells despite the altered polarity of basolateral proteins with tyrosine-based signals in this cell line. These results provide the first direct insights into how E-cadherin is targeted to the basolateral membrane.

Microtubule integrity is essential for apical polarization and epithelial morphogenesis in the thyroid.

In this study, we examined the contribution of microtubules to epithelial morphogenesis in primary thyroid cell cultures. Thyroid follicles consist of a single layer of polarized epithelial cells surrounding a closed compartment, the follicular lumen. Freshly isolated porcine thyroid cells aggregate and reorganize to form follicles when grown in primary cultures. Follicular reorganization is principally a morphogenetic process that entails the assembly of biochemically distinct apical and basolateral membrane domains, delimited by tight junctions. The establishment of cell surface polarity during folliculogenesis coincided with the polarized redistribution of microtubules, predominantly in the developing apical poles of cells. Disruption of microtubule integrity using either colchicine or nocodazole caused loss of defined apical membrane domains, tight junctions and follicular lumina. Apical membrane and tight junction markers became randomly distributed at the outer surfaces of aggregates. In contrast, the basolateral surface markers, E-cadherin and Na(+),K(+)-ATPase, remained correctly localized at sites of cell-cell contact and at the free surfaces of cell aggregates. These findings demonstrate that microtubules play a necessary role in thyroid epithelial morphogenesis. Specifically, microtubules are essential to preserve the correct localization of apical membrane components within enclosed cellular aggregates, a situation that is also likely to pertain where lumina must be formed from solid aggregates of epithelial precursors.

Recycling of E-cadherin: a potential mechanism for regulating cadherin dynamics.

E-Cadherin plays critical roles in many aspects of cell adhesion, epithelial development, and the establishment and maintenance of epithelial polarity. The fate of E-cadherin once it is delivered to the basolateral cell surface, and the mechanisms which govern its participation in adherens junctions, are not well understood. Using surface biotinylation and recycling assays, we observed that some of the cell surface E-cadherin is actively internalized and is then recycled back to the plasma membrane. The pool of E-cadherin undergoing endocytosis and recycling was markedly increased in cells without stable cell-cell contacts, i.e., in preconfluent cells and after cell contacts were disrupted by depletion of extracellular Ca2+, suggesting that endocytic trafficking of E-cadherin is regulated by cell-cell contact. The reformation of cell junctions after replacement of Ca2+ was then found to be inhibited when recycling of endocytosed E-cadherin was disrupted by bafilomycin treatment. The endocytosis and recycling of E-cadherin and of the transferrin receptor were similarly inhibited by potassium depletion and by bafilomycin treatment, and both proteins were accumulated in intracellular compartments by an 18 degrees C temperature block, suggesting that endocytosis may occur via a clathrin-mediated pathway. We conclude that a pool of surface E-cadherin is constantly trafficked through an endocytic, recycling pathway and that this may provide a mechanism for regulating the availability of E-cadherin for junction formation in development, tissue remodeling, and tumorigenesis.

The juxtamembrane region of the cadherin cytoplasmic tail supports lateral clustering, adhesive strengthening, and interaction with p120ctn.

Cadherin cell-cell adhesion molecules form membrane-spanning molecular complexes that couple homophilic binding by the cadherin ectodomain to the actin cytoskeleton. A fundamental issue in cadherin biology is how this complex converts the weak intrinsic binding activity of the ectodomain into strong adhesion. Recently we demonstrated that cellular cadherins cluster in a ligand-dependent fashion when cells attached to substrata coated with the adhesive ectodomain of Xenopus C-cadherin (CEC1-5). Moreover, forced clustering of the ectodomain alone significantly strengthened adhesiveness (Yap, A.S., W.M. Brieher, M. Pruschy, and B.M. Gumbiner. Curr. Biol. 7:308-315). In this study we sought to identify the determinants of the cadherin cytoplasmic tail responsible for clustering activity. A deletion mutant of C-cadherin (CT669) that retained the juxtamembrane 94-amino acid region of the cytoplasmic tail, but not the beta-catenin-binding domain, clustered upon attachment to substrata coated with CEC1-5. Like wild-type C-cadherin, this clustering was ligand dependent. In contrast, mutant molecules lacking either the complete cytoplasmic tail or just the juxtamembrane region did not cluster. The juxtamembrane region was itself sufficient to induce clustering when fused to a heterologous membrane-anchored protein, albeit in a ligand-independent fashion. The CT669 cadherin mutant also displayed significant adhesive activity when tested in laminar flow detachment assays and aggregation assays. Purification of proteins binding to the juxtamembrane region revealed that the major associated protein is p120(ctn). These findings identify the juxtamembrane region of the cadherin cytoplasmic tail as a functionally active region supporting cadherin clustering and adhesive strength and raise the possibility that p120(ctn) is involved in clustering and cell adhesion.

Protein tyrosine phosphorylation influences adhesive junction assembly and follicular organization of cultured thyroid epithelial cells.

The follicular histoarchitecture of the thyroid forms the anatomical basis for thyroid physiology and is commonly disturbed in diseases of the thyroid. We have used cultured porcine thyroid cells to study thyroid epithelial morphogenesis and its regulation. When cultured in the presence of TSH, freshly isolated thyroid cells reorganize to form follicles within three-dimensional cell aggregates. However, when established follicles are washed into TSH-free medium, thyroid cells spread and migrate to convert follicles into confluent epithelioid monolayers, activating morphogenetic mechanisms, such as cell locomotility, that may be relevant to thyroid inflammation and tumor invasiveness. The phenomenon of follicle to monolayer conversion, therefore, provides an opportunity to identify morphogenetic mechanisms that 1) must be tonically inhibited to maintain follicular organization and 2) may contribute to pathogenetic disturbances of follicular architecture when functioning aberrantly. In this study we found that follicle to monolayer conversion is associated with an increase in cellular phosphotyrosine. This was particularly evident at nascent focal adhesions (cell-substrate adhesive junctions) and later at cell-cell junctions. Focal adhesion assembly was accompanied by reorganization of the actin cytoskeleton, with the appearance of prominent stress fibers. Genistein, a potent inhibitor of protein tyrosine kinases, inhibited the accumulation of phosphotyrosine, focal adhesion assembly, and follicle to monolayer conversion. We conclude that tyrosine phosphorylation exerts an important influence on thyroid epithelial organization in culture, at least partly mediated through regulation of focal adhesion assembly.

Lateral clustering of the adhesive ectodomain: a fundamental determinant of cadherin function.

BACKGROUND: Classical cadherin-based cellular adhesion is mediated by a multicomponent protein complex that links the adhesive binding activity of the cadherin ectodomain to the actin cytoskeleton. Despite the importance of cadherins in morphogenesis and development, we know very little about how cells determine and alter cadherin adhesive strength. In this study, we sought to identify specific cellular mechanisms that modulate cadherin function by studying adhesion between cells transfected with Xenopus C-cadherin mutant molecules and substrata coated with the purified ectodomain of C-cadherin. RESULTS: Using the FKBP-FK1012 protein oligomerization system, we found that forced clustering, in cells, of cadherin mutants lacking the cytoplasmic tail significantly increased cellular adhesive strength. Therefore, redistribution of the adhesive binding sites of cells into clusters can influence adhesion independently of other protein interactions mediated by the cadherin cytoplasmic tail. Furthermore, cells transfected with full-length C-cadherin demonstrated dynamic changes in adhesion over time that correlated with clustering but not with changes in the surface expression of C-cadherin or in the composition of the cadherin-catenin complex. The cytoplasmic tail was, however, necessary for clustering of wild-type cadherin. CONCLUSIONS: These studies directly demonstrate a fundamental role for lateral clustering in cadherin function. The distribution of cadherin binding sites presented at the cell surface, a cellular property which is regulated by the cadherin cytoplasmic tail, is an important mechanism which modulates cellular adhesion independently of cytoskeletal activity or signalling.

Molecular and functional analysis of cadherin-based adherens junctions.

Adherens junctions are specialized forms of cadherin-based adhesive contacts important for tissue organization in developing and adult organisms. Cadherins form protein complexes with cytoplasmic proteins (catenins) that convert the specific, homophilic-binding capacity of the extracellular domain into stable cell adhesion. The extracellular domains of cadherins form parallel dimers that possess intrinsic homophilic-binding activity. Cytoplasmic interactions can influence the function of the ectodomain by a number of potential mechanisms, including redistribution of binding sites into clusters, providing cytoskeletal anchorage, and mediating physiological regulation of cadherin function. Adherens junctions are likely to serve specific, specialized functions beyond the basic adhesive process. These functions include coupling cytoskeletal force generation to strongly adherent sites on the cell surface and the regulation of intracellular signaling events.

Lateral dimerization is required for the homophilic binding activity of C-cadherin.

Regulation of cadherin-mediated adhesion can occur rapidly at the cell surface. To understand the mechanism underlying cadherin regulation, it is essential to elucidate the homophilic binding mechanism that underlies all cadherin-mediated functions. Therefore, we have investigated the structural and functional properties of the extracellular segment of Xenopus C-cadherin using a purified, recombinant protein (CEC 1-5). CEC 1-5 supported adhesion of CHO cells expressing C-cadherin. The extracellular segment was also capable of mediating aggregation of microspheres. Chemical cross-linking and gel filtration revealed that CEC 1-5 formed dimers in the presence as well as absence of calcium. Analysis of the functional activity of purified dimers and monomers demonstrated that dimers retained substantially greater homophilic binding activity than monomers. These results demonstrate that lateral dimerization is necessary for homophilic binding between cadherin extracellular segments and suggest multiple potential mechanisms for the regulation of cadherin activity. Since the extracellular segment alone possessed significant homophilic binding activity, the adhesive activity of the extracellular segment in a cellular context was analyzed. The adhesion of CHO cells expressing a truncated version of C-cadherin lacking the cytoplasmic tail was compared to cells expressing the wild-type C-cadherin using a laminar flow assay on substrates coated with CEC 1-5. CHO cells expressing the truncated C-cadherin were able to attach to CEC 1-5 and to resist detachment by low shear forces, demonstrating that tailless C-cadherin can mediate basic, weak adhesion of CHO cells. However, cells expressing the truncated C-cadherin did not exhibit the complete adhesive activity of cells expressing wild-type C-cadherin. Cells expressing wild-type C-cadherin remained attached to CEC 1-5 at high shear forces, while cells expressing the tailless C-cadherin did not adhere well at high shear forces. These results suggest that there may be two states of cadherin-mediated adhesion. The first, relatively weak state can be mediated through interactions between the extracellular segments alone. The second strong adhesive state is critically dependent on the cytoplasmic tail.

Cadherin-mediated adhesion and apical membrane assembly define distinct steps during thyroid epithelial polarization and lumen formation.

The biogenesis of follicles from aggregates of precursor cells is an important morphogenetic process in thyroid embryology. It necessitates the creation of a polarized cell phenotype, assembly of specialized cell-cell junctions, and generation of follicular lumena. In this study we sought to investigate the relationship between cell polarization and lumen formation by studying the cell surface events that occurred when freshly isolated adult porcine thyroid cells reorganized to form follicles in primary culture. Follicular reorganization entailed the initial formation of solid three-dimensional cell aggregates and the subsequent appearance of lumena within aggregates. During the initial stage of cell aggregation, the adhesion molecule, E-cadherin, became expressed at all surfaces involved in cell-cell contact. Aggregation was inhibited by monoclonal antibodies that block cadherin function, indicating directly that E-cadherin is a dominant initial cell-cell adhesion molecule. Cell aggregation was also associated with the recruitment to the cell surface of ZO-1, a tight junction-associated protein, and Na+/K(+)-adenosine triphosphatase. These proteins were initially found throughout regions of cell-cell contact and only subsequently redistributed to their mature locations in tight junctions and the basolateral cell surface, respectively. In contrast, components associated with the apical membrane were first detected within large intracellular vacuoles, which subsequently fused with the cell surface between maturing tight junctions to yield the apical membrane domain and nascent follicular lumena. Follicle formation occurred independently of basal lamina assembly and TSH, although maintenance of follicular architecture required the presence of this hormone. These findings indicate that cultured follicles form in two distinct stages: 1) initial aggregation mediated by E-cadherin and associated with recruitment of components of both tight junctions and the basolateral membrane domain, and 2) subsequent formation of a specialized apical membrane domain by coordinated fusion of intracellular vacuoles at sites of the cell surface where tight junctions are maturing. We propose that follicular morphogenesis may arise as a consequence of epithelial cell polarization within coherent three-dimensional cell aggregates.

Microtubule integrity is necessary for the epithelial barrier function of cultured thyroid cell monolayers.

Vectorial transport in the thyroid epithelium requires an efficient barrier against passive paracellular flux, a role which is principally performed by the tight junction (zonula occludens). There is increasing evidence that tight junction integrity is determined by integral and peripheral membrane proteins which interact with the cell cytoskeleton. Although the contribution of the actin cytoskeleton to tight junction physiology has been intensively studied, less is known about possible interactions with microtubules. In the present study we used electrophysiological and immunohistochemical approaches to investigate the contribution of microtubules to the paracellular barrier in cultured thyroid cell monolayers which displayed a high transepithelial electrical resistance (6000-9000 ohm.cm2). Colchicine (1 microM) caused a progressive fall in electrical resistance to < 10% of baseline after 6 h and depolarization of the transepithelial electrical potential difference consistent with a significant increase in paracellular permeability. The effect of colchicine on TER was not affected by agents which inhibit the major apical conductances of thyroid cells but was reversed upon removal of the drug. Immunofluorescent staining for tubulin combined with confocal laser scanning microscopy demonstrated that thyroid cells possessed a dense microtubule network extending throughout the cytoplasm which was destroyed by colchicine. Colchicine also produced changes in the localization of the tight junction-associated protein, ZO-1: its normally continuous junctional distribution was disrupted by striking discontinuities and the appearance of many fine strands which extended into the cytoplasm. A similar disruption in E-cadherin staining was also observed, but colchicine did not affect the distribution of vinculin associated with adherens junctions nor the integrity of the perijunctional actin ring. We conclude that microtubules are necessary for the functional and structural integrity of tight junctions in this electrically tight, transporting epithelium.

Characterization of cell polarity and epithelial junctions in the choriocarcinoma cell line, JAR.

In the placenta the trophoblast cell layer separates maternal and fetal circulations and is involved in the active transport of selected substances across this barrier. We have used the JAR choriocarcinoma cell line to study aspects of trophoblast membrane transport. To determine whether JAR cells could be used in studies of vectorial transepithelial transport it was necessary to determine whether these cells were polarized and assembled tight junctions. In the present study we investigated JAR cells using a range of markers for specific cell surface domains combined with confocal laser scanning microscopy. Freshly isolated cells initially formed a confluent epithelial monolayer with recruitment of a tight junction-associated protein, ZO-1, and a cell adhesion molecule, E-cadherin, to the surface at sites of cell-cell contact. They did not, however, display cell surface polarization, as NaK-ATPase was not segregated in the basolateral domain, and a differentiated apical cell surface was not assembled. The monolayer stage was also unstable, as continued proliferation resulted in the formation of multilayered aggregates where ZO-1 and E-cadherin were lost from the cell surface. These results suggest that the JAR cell line is unlikely to be a suitable model for studies of transepithelial transport in the placenta.

Vinculin localization and actin stress fibers differ in thyroid cells organized as monolayers or follicles.

In epithelial cells interactions between the actin cytoskeleton and cell-cell junctions regulate paracellular permeability and participate in morphogenesis. We have studied the relationship between supracellular morphology and actin-junction interactions using primary cultures of porcine thyroid cells grown either as three-dimensional follicles or as open monolayers. Regardless of morphology, thyroid cells assembled occluding and adhesive junctions containing ZO-1 and E-cadherin, respectively, and showed F-actin staining in apical microvilli and a perijunctional ring. In monolayers, actin stress fibers were also observed in the apical and basal poles of cells, where they terminated in the vinculin-rich zonula adherens and in cell-substrate focal adhesions, respectively. Surprisingly, we were unable to detect vinculin localization in follicular cells, which also did not form stress fibers. Immunoblotting confirmed significantly greater vinculin in triton-insoluble fractions from monolayer cells compared with follicular cells. Incubation of monolayers with 8 chloro(phenylthio)-cyclic AMP decreased the level of immunodetectable vinculin in the zonula adherens, indicating that junctional incorporation of vinculin was regulated by cyclic AMP. In monolayer cultures, cytochalasin D (1 microM) cause actin filaments to aggregate associated with retraction of cells from one another and the disruption of cell junctions. Despite morphologically similar perturbations of actin organization in follicular cultures treated with cytochalasin D, junctional staining of ZO-1 and E-cadherin was preserved and cells remained adherent to one another. We conclude that in cultured thyroid cells structural and functional associations between actin filaments and cellular junctions differ depending upon the supracellular morphology in which cells are grown. One important underlying mechanism appears to be regulation of vinculin incorporation into adhesive junctions by cyclic AMP.

Thyrotropin inhibits the intrinsic locomotility of thyroid cells organized as follicles in primary culture.

The regulation of cell locomotion is a fundamental determinant of tissue architecture. Even in solid tissues of adult organisms cells often retain an intrinsic locomotor capacity which is activated during wound healing or tumor metastasis. In this study we have examined the role of cell locomotion in an in vitro model of thyroid epithelial pattern generation. Primary cultures of adult porcine thyroid cells reorganize to form follicles within three-dimensional cell aggregates when stimulated by thyrotropin (thyroid-stimulating hormone, TSH). Removal of TSH from the culture medium caused established follicles to reorganize into a confluent, two-dimensional epithelioid monolayer. The earliest observed change was the appearance of spreading cells at the peripheries of aggregates. These cells displayed broad lamellipodia whose formation was associated with the redistribution of microfilaments and microtubules and the accumulation of myosin. Spreading cells could migrate into, and fill, artificial wounds several millimeters wide without evidence of cell proliferation, indicating that cells became locomotile as they spread from follicles to form monolayer. Both spreading and migration were inhibited by cytochalasin B. In contrast, cells spread in the presence of colchine, but failed to migrate subsequently. Thyroid cell locomotility from follicles was inhibited by TSH, a cAMP analog, and a cell-free membrane fraction. However, migration from established monolayer cultures was not affected by these regulatory agents. This indicated that cell spreading was an important regulatory locus in thyroid cell patterning. We conclude that the tonic inhibition of thyroid cell locomotility contributes to the maintenance of follicular architecture in vitro. TSH and cell-cell contact may inhibit locomotion by preventing follicular cells from spreading, the earliest step in the morphogenetic conversion of follicles to monolayer.

Thyroid epithelial morphogenesis in vitro: a role for bumetanide-sensitive Cl- secretion during follicular lumen development.

The structural and functional unit of the thyroid gland is the follicle, consisting of a closed lumen surrounded by a single layer of polarized epithelial cells. In this paper we have attempted to characterize the process of lumenal development when primary cultures of porcine thyroid cells reorganized to form follicles. Cells incubated with the loop diuretic, bumetanide, an inhibitor of NaK2Cl cotransport, aggregated but failed to form normal follicles. Laser scanning confocal microscopy combined with immunohistochemical markers of thyroid cell-surface proteins demonstrated that in the presence of bumetanide cells polarized and assembled ZO-1-containing tight junctions separating their apical and basolateral membrane domains. Cultures formed small lumena but their subsequent growth was inhibited by bumetanide. Electrophysiological studies confirmed that bumetanide-sensitive Cl- transport was the major contributor to the transepithelial electrical potential difference across the follicular wall after 48 h incubation. Other potential mechanisms did not contribute significantly to follicular lumenal growth. In particular, bumetanide did not affect cell proliferation and, in contrast to tissue follicles, thyroglobulin could not be detected within the lumena of cultured follicles. We conclude that thyroid follicular reorganization involves two distinct and separate phases of lumenal development: initial lumen formation which probably reflects the assembly of a specialized apical membrane domain; and subsequent lumenal growth which is mediated by the inward transport of Cl- by polarized epithelial cells.