Fibroblastic Shape Research Articles

Keratocan Expression of Murine Keratocytes Is Maintained on Amniotic Membrane by Down-regulating Transforming Growth Factor-β Signaling

Keratocytes in the corneal stroma express keratan sulfate-containing proteoglycans including cornea-specific keratocan. On plastic dishes, human, bovine, and rabbit keratocytes lose their characteristic dendritic morphology and keratocan expression when cultured in serum-containing media. Herein, we demonstrated that murine keratocytes also acquired a fibroblastic shape and lost keratocan expression after first passage when cultured on plastic in the presence of serum. In contrast, cells expanded on human amniotic membrane (AM) stromal matrix maintained a three-dimensional dendritic morphology and expressed keratocan mRNA and protein for at least 8 passages before senescence. When keratocytes were cultured on AM, the promoter activity of transforming growth factor (TGF)-beta2 and TGF-beta receptor II was down-regulated as compared with that on plastic. Furthermore, cells on AM continuously retained Smad 2 and Smad 4 in the cytoplasm and did not express alpha-smooth muscle actin, even when 10 ng/ml TGF-beta1 was added in a serum-free medium for up to 5 days. In parallel to such down-regulation of TGF-beta signaling, keratocan promoter-driven ECFP expression was observed in cells cultured either on AM in the presence of serum or on plastic containing serum treated with a neutralizing antibody to TGF-beta. Collectively, these results indicate that down-regulation of Smad-mediated TGF-beta signaling is an important mechanism for cultured keratocytes to maintain a normal phenotype while continuously expanded in a serum-containing medium. This strategy of suppressing TGF-beta signaling, achieved by AM stromal matrix in part via suppression of TGF-beta gene transcription, can be used to expand keratocytes in culture without the use of AM in the future.

Effects of IκB kinase α on the differentiation of squamous carcinoma cells

IkappaB kinase (IKK) alpha and beta share the function to phosphorylate IkappaB to activate a transcription factor NF-kappaB. Recent reports, however, revealed differences in the functions of the two kinases. The present study was designed to determine a unique function of IKKalpha on the differentiation of squamous cell carcinoma (SCC). Transfection with IKKalpha gene, but neither IKKbeta nor NF-kappaB gene, inhibited the constitutive expressions as well as extracellular calcium-induced expressions of involucrin and filaggrin, epithelial differentiation markers, in cultured SCC cells. Morphological changes from polygonal to fibroblastic shape were seen in the SCC cells stably expressing green-fluorescent protein (GFP)-fused IKKalpha while intracellular localization of GFP-IKKalpha differed from that of GFP-IKKbeta. Interestingly, phorbol myristate acetate together with IKKalpha gene transfection strongly inhibited the expression of involucrin in SCC cells and induced the phosphorylation of serine residue in IKKalpha, suggesting that protein kinase C is involved in the effect of IKKalpha on the differentiation of SCC cells. In conclusion, high expression of IKKalpha may serve as an intracellular signal to halt the epithelial differentiation of SCC cells.

An in Vitro Approach to the Chronological Aging of Skin by Glycation of the Collagen: The Biological Effect of Glycation on the Reconstructed Skin Model

Glycation is a slow, nonenzymatic reaction that takes place between free amino groups in proteins primarily from lysine and a reducing sugar such as glucose or ribose. In skin, this reaction creates new residues or formations of cross-links (advanced glycation end products, AGEs) in the extracellular matrix of the dermis. The formation of these bridges between dermal molecules is supposed to be responsible for loss of elasticity or other properties of the dermis observed during aging. Glycation may therefore play an important role in chronologic aging. In order to examine this hypothesis, we have developed a reconstructed skin model made of a modified dermal compartment that is a fibroblast-contracted collagen lattice prepared with preglycated collagen. The presence of AGEs (glycoxidation products) in the skin equivalents was evidenced using specific antibodies against carboxymethyllysine (CML). Several changes were observed after collagen glycation: (1) fibroblast shape and distribution (vimentin staining) were modified; (2) extracellular matrix molecules and the dermal-epidermal junction zone seemed to be enhanced (procollagen I and III, collagen IV and VII stainings); (3) stainings for beta1 and alpha6 integrins were also increased in the epidermal cell layer; and (4) collagenase activity was increased. To verify the biological effect of glycation, we used the well-known glycation inhibitor aminoguanidine. After aminoguanidine treatment, we found a low CML amount and decreased distribution of markers previously overexpressed in glycated skin constructs. These in vitro findings were at least in part related to aging in vivo and demonstrate an actual effect of glycation in skin aging.

Characterization of renal interstitial fibroblast-specific protein 1/S100A4-positive cells in healthy and inflamed rodent kidneys

Fibrosis is considered as a central factor in the loss of renal function in chronic kidney diseases. The origin of fibroblasts and myofibroblasts that accumulate in the interstitium of the diseased kidney is still a matter of debate. It has been shown that accumulation of myofibroblasts in inflamed and fibrotic kidneys is associated with upregulation of fibroblast-specific protein 1 (FSP1, S100A4), not only in the renal interstitium but also in the injured renal epithelia. The tubular expression of FSP1 has been taken as evidence of myofibroblast formation by epithelial-mesenchymal transition (EMT). The identity of FSP1/S100A4 cells has not been defined in detail. We originally intended to use FSP1/S100A4 as a marker of putative EMT in a model of distal tubular injury. However, since the immunoreactivity of FSP1 did not seem to fit with the distribution and shape of fibroblasts or myofibroblasts, we undertook the characterization of FSP1/S100A4-expressing cells in the interstitium of rodent kidneys. We performed immunolabeling for FSP1/S100A4 on thin cryostat sections of perfusion-fixed rat and mouse kidneys with peritubular inflammation, induced by thiazides and glomerulonephritis, respectively, in combination with ecto-5'-nucleotidase (5'NT), recognizing local cortical peritubular fibroblasts, with CD45, MHC class II, CD3, CD4 and Thy 1, recognizing mononuclear cells, with alpha smooth muscle actin (alphaSMA), as marker for myofibroblasts, and vimentin for intracellular intermediate filaments in cells of mesenchymal origin. In the healthy interstitium of rodents the rare FSP1/S100A4+ cells consistently co-expressed CD45 or lymphocyte surface molecules. Around the injured distal tubules of rats treated for 3-4 days with thiazides, FSP1+/S100A4+, 5'NT+, alphaSMA+, CD45+ and MHC class II+ cells accumulated. FSP1+/S100A4+ cells consistently co-expressed CD45. In the inflamed regions, alphaSMA was co-expressed by 5'NT+ cells. In glomerulonephritic mice, FSP1+/S100A4+ cells co-expressed Thy 1, CD4 or CD3. Thus, in the inflamed interstitium around distal tubules of rats and of glomerulonephritic mice, the majority of FSP1+ cells express markers of mononuclear cells. Consequently, the usefulness of FSP1/S100A4 as a tool for detection of (myo)fibroblasts in inflamed kidneys and of EMT in vivo is put into question. In the given rat model the consistent co-expression of alphaSMA and 5'NT suggests that myofibroblasts originate from resident peritubular fibroblasts.

Expression of growth-factor Receptors in Rat Socket coagulum-derived Cells

The purpose of this study was to determine if periodontal ligament (PDL) fibroblasts are capable of expressing growth factor receptors during their differentiation. PDL cells, obtained from 2-day-old coagulum in the tooth socket of Sprague-Dawley rats after tooth extraction, were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and antibiotics. Confluent cells were grown in the presence of 50 μg/ml ascorbic acid (AA), 10mM β-glycerophosphate (βGP), and 5 µM dexamethasone (Dex) for up to 3 weeks. Morphological changes in PDL cells during the 3-week cultures in the presence of AA, βGP, and Dex were investigated under a phase-contrast microscope. The messenger ribonucleic acid (mRNA) expression pattern of growth factors, including platelet-derived growth factor receptor-alpha and beta (PDGF-Rα and PDGFRβ), insulin-like growth factor type 1 and 2 receptors (IGF-1R and IGF-2R), and fibroblast growth factor receptor-4 (FGF-R4), were investigated in PDL cells, using Northern blot analysis. Based on the appearance of PDL cell cultures under the phase-contrast microscope, 4 morphologically distinct stages were identified: the confluent stage, the multilayer stage, the nodule stage and the mineralization stage. In the confluent stage (stage Ⅰ), PDL cells had a fibroblast-like or polygonal shape and formed a monolayer. On the 7(superscript th) day after treatment with AA, βGP, and Dex (stage Ⅱ), cell proliferation occurred in focal areas generating multilayered cells with a fibroblastic shape. After the 14(superscript th) day (stage Ⅲ) the multilayered cells formed round nodules by depositing a matrix between the cell layers. Heavy mineralization of the matrix was first seen on the 21st day after mineralized medium treatment and was characterized by the presence of refringent material covering the nodules, referred to as the mineralization stage (stage Ⅳ). By Northern blot analysis, it was determined that PDGF-Rα was weakly expressed in the confluent stage and then increased with the progression of PDL cell differentiation, while the level of PDGF-Rβ transcripts remained high throughout the 4 stages. IGF-1R gene activities were also high throughout the four stages. In contrast, expressions of IGF-2R and FGF-R4 mRNA were undetectable throughout differentiation. These results indicate that growth factor receptors are differentially expressed by PDL cells, which may be involved in the regulation of growth and differentiation of PDL cells.

Biodegradable microspheres for prolidase delivery to human cultured fibroblasts.

Prolidase deficiency (PD) is a rare autosomal recessive disorder caused by inadequate levels of the cytosolic exopeptidase prolidase (E.C. 3.4.13.9), for which there is not, as yet, a resolutive cure. We have investigated whether biodegradable microspheres loaded with prolidase could release active enzyme inside cells, to consider this system as a possible therapeutic approach for prolidase deficiency. Poly(lactide-co-glycolide) microspheres were prepared, modifying the classical double emulsion solvent evaporation method to mitigate the burst effect of the enzyme from the microspheres. Ex-vivo experiments were performed, by incubating microencapsulated prolidase with cultured fibroblasts from PD patients and from controls, to determine the amount of active enzyme delivered to the cells. The microparticulate drug delivery system described carried small amounts of active prolidase inside fibroblasts, ensuring a response to the intracellular accumulation of X-Pro dipeptides, the mechanism that is supposed to be responsible for the development of clinical manifestations of this disorder in man. A positive result of the presence of active enzyme inside cells was an improvement in fibroblast shape.

514. Potential of Bone Marrow Mesenchymal Stem Cells for Cystic Fibrosis Therapy

Bone marrow mesenchymal stem cells (MSCs) have been shown to differentiate into pulmonary epithelial cells in animal models. To explore whether human MSCs can undergo differentiation into airway epithelia, which can be exploited as a novel gene therapy strategy for cystic fibrosis (CF), human MSCs expressing green florescent protein (GFP) were mixed with primary human airway epithelial cells in a ratio of 1:5, 1:10, or 1:20, and seeded on a semi-permeable filter membrane coated with human placenta collagen type VI. The cells were cultured at an air-liquid interface and were able to maintain the apical surface dry after one to two weeks with transepithelial resistance greater than 700 Ohms, suggesting the formation of tight junctions. Immunofluorescent staining with an antibody against cytokeratin 18 (CK18) demonstrated that the stem cells co-cultured with human airway cells expressed this epithelium-specific marker, whereas prior to the culture, there is no detectable CK18 expression by this approach. Morphologically, the GFP-labeled stem cells converted their original thin-spindle fibroblast shape or flat polygonal cell shape to a typical epithelial cuboidal shape with an accessory structure like cilia by confocal microscopy, suggesting that the hMSCs in the co-culture system transdifferentiate into airway epithelial cells. To confirm the finding, we further did immunostaining with an antibody against occludin, an epithelial tight junction protein. Flow cytometry and fluorescent microscopy showed that ~10% of the initially loaded GFP-MSCs express this critical epithelial marker. By cell sorting, we isolated the co-cultured hMSCs, and RT-PCR was performed with primers specific to human CFTR. The results demonstrated that the air-liquid culture induces hMSCs to express the CFTR gene. Furthermore, co-culture of normal hMSCs with CF airway epithelial cells resulted in the wild-type CFTR expression. More importantly, the MSCs isolated from CF patients were able to be gene-corrected. In vitro differentiation assays proved that the CFTR gene-corrected CF MSCs still possess their naive multipotency, such as differentiation along osteogenic, adipogenic, and chondrogenic pathways. The gene-corrected CF MSCs were able to contribute to apical Cl- secretion by chloride efflux assay in response to cAMP agonists. Conditions to increase the efficacy of MSC differentiation are currently under investigation. Therefore, we conclude that bone marrow mesenchymal stem cells may have potential applications in treating human airway diseases, such as CF.

Biglycan and decorin induce morphological and cytoskeletal changes involving signalling by the small GTPases RhoA and Rac1 resulting in lung fibroblast migration.

Biglycan and decorin are small chondroitin/dermatan sulphate proteoglycans in the extracellular matrix of connective tissue that belong to the family of structurally related proteoglycans called small leucine-rich repeat proteins. We show for the first time that biglycan and decorin induce morphological and cytoskeletal changes in fibroblasts, resulting in an increase in migration. Biglycan changed the cell shape of fibroblasts with formation of long protruding filamentous processes. This was also seen for decorin but to a lesser extent. Using fluorescence staining of F-actin fibres it was possible to show that these long filamentous processes were supported by long thick bundles of actin, together with an induced formation of stress fibres after stimulation with biglycan and decorin. Moreover, a reorganisation of alpha-smooth muscle actin was clearly seen in these cultures. Decorin also stimulated alpha-smooth muscle actin expression in the cells. Using cDNA Atlas Arrays we were also able to show that the mRNA level of a number of the intracellular regulators and effectors involved in cell migration were increased. For example, the focal adhesion proteins paxillin and zyxin, and some of the small Rho GTPases such as RhoA, Rac1 and Cdc42 were upregulated. After treatment with biglycan or decorin, additional results showed an increased activation of RhoA (1.8- and 1.5-fold, respectively) and Rac1 (1.8- and 1.5-fold, respectively) after 15 minutes. These factors are known to be involved in fibroblast migration, and as expected a 1.3- to 1.6-fold increase in migration could be observed after stimulation with biglycan or decorin. This induced migration was caused by the core protein, as treatment with glycosaminoglycan chains alone did not have any effect. In summary, these data indicate that biglycan- and decorin-induced fibroblast cytoskeletal and signalling changes result in an increased cell migration, and demonstrate their potential role in the remodelling process.

LDLs induce fibroblast spreading independently of the LDL receptor via activation of the p38 MAPK pathway

Because adventitial fibroblasts play an important role in the repair of blood vessels, we assessed whether elevation in LDL concentrations would affect fibroblast function and whether this depended on activation of intracellular signaling pathways. We show here that in primary human fibroblasts, LDLs induced transient activation of the p38 mitogen-activated protein kinase (MAPK) pathway, but not the c-Jun N-terminal kinase MAPK pathway. This activation did not require the recruitment of the LDL receptor (LDLR), because LDLs efficiently stimulated the p38 MAPK pathway in human and mouse fibroblasts lacking functional LDLR, and because receptor-associated protein, an LDLR family antagonist, did not block the LDL-induced p38 activation. LDL particles also induced lamellipodia formation and cell spreading. These effects were blocked by SB203580, a specific p38 inhibitor. Our data demonstrate that LDLs can regulate the shape of fibroblasts in a p38 MAPK-dependent manner, a mechanism that may participate in wound healing or vessel remodeling as in atherosclerosis.

Cell–matrix interactions of in vitro human skin fibroblasts upon addition of hyaluronan

Normal human skin fibroblasts were grown in a three-dimensional collagen gel or in monolayer in the presence or absence of high molecular weight hyaluronan (HA) to assess the influence of extracellular HA on cell–matrix interactions. HA incorporated into the collagen gel or added to the culture medium did not modify lattice retraction with time. The effect was independent from HA molecular weight (from 7.5×10 5 to 2.7×10 6 Da) and concentration (from 0.1 up to 1 mg/ml). HA did not affect shape and distribution of fibroblasts within the gel, whereas it induced the actin filaments to organise into thicker cables running underneath the plasma membrane. The same phenomenon was observed in fibroblasts grown in monolayer. By contrast, vimentin cytoskeleton and cell–substrate focal adhesions were not modified by exogenous HA. The number of fibroblasts attached to HA-coated dishes was always significantly lower compared to plastic and to collagen type I-coated plates. By contrast, adhesion was not affected by soluble HA added to the medium nor by anti-CD44 and anti-RHAMM-IHABP polyclonals. After 24-h seeding on collagen type I or on plastic, cells were large and spread. Conversely, cells adherent to HA-coated surfaces were long, thin and aligned into rows; alcian blue showed that cells were attached to the plastic in between HA bundles. Therefore, normal human skin fibroblasts exhibit very scarce, if any, adhesion to matrix HA, either soluble or immobilised. Moreover, even at high concentration, HA molecules do not exert any visco-mechanical effect on lattice retraction and do not interfere with fibroblast–collagen interactions nor with focal adhesion contacts of fibroblasts with the substrate. This is probably relevant in organogenesis and wound repair. By contrast, HA greatly modifies the organisation of the actin cytoskeleton, suggesting that CD44-mediated signal transduction by HA may affect cell locomotion and orientation, as indicated by the fusiform shape of fibroblasts grown in the presence of immobilised HA. A role of HA in cell orientation could be relevant for the deposition of collagen fibrils in regeneration and tissue remodelling.

Mechanisms of polarization of the shape of fibroblasts and epitheliocytes: Separation of the roles of microtubules and Rho-dependent actin-myosin contractility.

Cultured fibroblasts possess a characteristic polarized phenotype manifested by an elongate cell body with an anterior lamella whose cell edge is divided into protrusion-forming and inactive zones. Disruption of the fibroblast microtubule cytoskeleton leads to an increase in Rho-dependent acto-myosin contractile activity and concomitant loss of structural polarity. The functional relationship of myosin-driven contractile activity to loss of fibroblast anterior-posterior polarity is unknown. To dissect the roles of microtubule assembly and of Rho-dependent contractility on structural polarization of cells, polarized fibroblasts and nonpolarized epitheliocytes were treated with the microtubule-depolymerizing drug, nocodazole, and/or the Rho kinase inhibitor, Y-27632. Fibroblasts incubated with Y-27632 increased their degree of polarization by developing a highly elongate cell body with multiple narrow processes extended from the edges of the cell. Treatment of fibroblasts with nocodazole, alone or in combination with Rho kinase inhibitor, produced discoid or polygonal cells having broad, flattened lamellae that did not form long lamellar extensions. Single cultured epitheliocytes of the IAR-2 line do not display anterior-posterior polarization. When treated with Y-27632, the cells acquired a polarized, elongate shape with narrow protrusions and wide lamellas. Nocodazole alone or in combination with Y-27632 did not change the discoid shape of epitheliocytes, however treatment with Y-27632 produced thinning of the lamellar cytoplasm. We conclude that microtubules provide the necessary framework for polarization of fibroblasts and epitheliocytes, whereas Rho-regulated contractility modulates the degree of polarization of fibroblasts and completely inhibits polarization in epitheliocytes.

FosB gene products trigger cell proliferation and morphological alteration with an increased expression of a novel processed form of galectin-1 in the rat 3Y1 embryo cell line.

In this study, we established rat 3Y1 embryo cell lines expressing FosB and DeltaFosB as fusion proteins (ER-FosB, ER-DeltaFosB) with the ligand-binding domain of human estrogen receptor (ER). The binding of estrogen to the fusion proteins resulted in their nuclear translocation. After estrogen administration, exponentially growing cells expressing ER-DeltaFosB, and to a lesser extent ER-FosB, underwent morphological alteration from the flat fibroblastic shape to an extended bipolar shape, and ceased proliferating. Such morphological alteration was also induced in quiescent cells expressing ER-DeltaFosB and ER-FosB after one round of cell division triggered by estrogen administration. The cells expressing ER-DeltaFosB changed shape frequently, and the content of F-actin in the cytoplasm detected by binding of Alexa 488-phalloidin significantly decreased after the morphological alteration. By two-dimensional gel electrophoresis analysis of cellular proteins from the cells expressing ER-DeltaFosB, we identified several proteins whose expression either increased or decreased after estrogen administration. Two of these proteins were identified from their amino acid sequences as novel processed form of galectin-1.

Abnormal accumulation of hyaluronan matrix diminishes contact inhibition of cell growth and promotes cell migration

Elevated hyaluronan biosynthesis and matrix deposition correlates with cell proliferation and migration. We ectopically expressed three isoforms of hyaluronan synthase (HAS1, HAS2, or HAS3) in nontransformed rat 3Y1 cells and observed a de novo, massive formation of a hyaluronan matrix that resulted in a partial loss of contact-mediated inhibition of cell growth and migration. All three HAS transfectants showed an enhanced motility in scratch wound assays, and a significant increase in their confluent cell densities. In high-density cultures, the HAS transfectants had a fibroblastic cell shape and markedly formed overlapping cell layers. This phenotype was more pronounced in the HAS2 transfectants than HAS1 or HAS3 transfectants, and occurred with significant alterations in the microfilament organization and N-cadherin distribution at the cell-cell border. Inhibition of a phosphatidylinositol 3-kinase (PI3-kinase) pathway resulted in reacquisition of the normal phenotype of HAS2 transfectants, suggesting that the intracellular PI3-kinase signaling regulates diminution of contact inhibition induced by formation of the massive hyaluronan matrix. Our observations suggest that hyaluronan and its matrix can modulate contact inhibition of cell growth and migration, and provide evidence for functional differences between hyaluronan synthesized by the different HAS proteins.

Use of Ti-coated replicas to investigate the effects on fibroblast shape of surfaces with varying roughness and constant chemical composition.

A two-stage replica technique with a subsequent titanium (Ti)-coating treatment was used to faithfully replicate topographies of polished, acid-etched, machined-like, finely blasted, coarsely blasted, coarsely blasted and acid-etched, and Ti plasma-sprayed Ti surfaces. The replicas were used to study the influence of different rough surface topographies on the response of human fibroblasts in vitro under conditions of constant surface chemistry for all surfaces. The surface topographies of the replicas were characterized using non-contact laser profilometry, scanning electron microscopy (SEM), and stereo-SEM, whereas surface chemistry was examined using X-ray photoelectron spectroscopy. Fibroblasts were trypsinized and plated onto the Ti-coated epoxy-resin replica surfaces for 24 h and observed with SEM. Fluorescein-5-thiosemicarbazide was used to stain the cell components including cell membrane, and the stained cells were optically sectioned using epifluorescent microscopy. The optical sections were computationally reconstructed to obtain three-dimensional images and cell volume and cell thickness determined. The different surface topographies were found to alter cell thickness and cell morphology. However, cell volume as computed from three-dimensional reconstructions was not affected by surface features. The results suggest that cells distort themselves to accommodate to rough surfaces but their volume is not significantly altered.

Rat embryo fibroblasts require both the cell-binding and the heparin-binding domains of fibronectin for survival

Fibronectin (FN) is known to transduce signal(s) to rescue cells from detachment-induced apoptosis (anoikis) through an integrin-mediated survival pathway. However, the functions of individual FN domains have not been studied in detail. In the present study we investigated whether the interaction of the cell-binding domain of FN with integrin is sufficient to rescue rat embryo fibroblasts (REFs) from detachment-induced apoptosis. REFs attached and spread normally after plating on substrates coated with either intact FN or a FN fragment, FN120, that contains the cell-binding domain but lacks the C-terminal heparin-binding domain, HepII. REFs on FN maintained a well-spread fibroblastic shape and even proliferated in serum-free medium at 20h after plating. In contrast, previously well-spread REFs on FN120 started losing fibroblastic shape with time and detached from FN120-coated plates after approx. 8h. Nuclear condensation indicated apototic cell death. This was due to the decreased activity/stability of focal adhesion kinase (pp125FAK) in the absence of HepII domain. A peptide in the HepII domain [peptide V, WQPPRARI (single-letter amino acid codes)], which has previously been implicated in cytoskeletal organization, rescued apoptotic changes. Consistently, pp125FAK phosphorylation was increased, and both cleavage of pp125FAK and activation of caspase 3 on FN120 were partly blocked by peptide V. Thus the interaction of the cell-binding domain with integrin has a major role in cell survival but is itself not sufficient for cell survival. One or more additional survival signals come from the HepII domain to regulate pp125FAK activity/stability.

Rat embryo fibroblasts require both the cell-binding and the heparin-binding domains of fibronectin for survival.

Fibronectin (FN) is known to transduce signal(s) to rescue cells from detachment-induced apoptosis (anoikis) through an integrin-mediated survival pathway. However, the functions of individual FN domains have not been studied in detail. In the present study we investigated whether the interaction of the cell-binding domain of FN with integrin is sufficient to rescue rat embryo fibroblasts (REFs) from detachment-induced apoptosis. REFs attached and spread normally after plating on substrates coated with either intact FN or a FN fragment, FN120, that contains the cell-binding domain but lacks the C-terminal heparin-binding domain, HepII. REFs on FN maintained a well-spread fibroblastic shape and even proliferated in serum-free medium at 20 h after plating. In contrast, previously well-spread REFs on FN120 started losing fibroblastic shape with time and detached from FN120-coated plates after approx. 8 h. Nuclear condensation indicated apototic cell death. This was due to the decreased activity/stability of focal adhesion kinase (pp125FAK) in the absence of HepII domain. A peptide in the HepII domain [peptide V, WQPPRARI (single-letter amino acid codes)], which has previously been implicated in cytoskeletal organization, rescued apoptotic changes. Consistently, pp125FAK phosphorylation was increased, and both cleavage of pp125FAK and activation of caspase 3 on FN120 were partly blocked by peptide V. Thus the interaction of the cell-binding domain with integrin has a major role in cell survival but is itself not sufficient for cell survival. One or more additional survival signals come from the HepII domain to regulate pp125FAK activity/stability.

Epitheliomesenchymal Transdifferentiation of Cultured RPE Cells

Retinal pigment epithelium (RPE) cells of the proliferative vitreoretinopathy (PVR) membrane take on the shape of fibroblasts and participate in fibrosis, thus deviating from the character of epithelial cells. This study was undertaken to evaluate RPE cell transdifferentiation in vitro. During the culture of porcine RPE cells, primary and 10th-passaged RPE cells were investigated for cell growth in response to transforming growth factor (TGF) β₂, change of phenotype and amount in collagen synthesis as well as expression of α-smooth-muscle actin (α-SMA). TGF-β₂ inhibited the proliferation of the primary cultures of RPE cells in a dose-dependent manner, while the spindle-shaped 10th-passaged RPE cells were not inhibited by TGF-β₂. The 10th-subcultured cells did not show much difference in the quality of collagen synthesis, other than type VIII collagen which was not produced. Collagen synthesis was dose-dependently stimulated by TGF-β₂. The stimulation by TGF-β₂ in the 10th-passaged RPE cells was much greater than in primary RPE cells. The 10th-subcultured RPE cells produced substantial α-SMA compared to α-SMA production by primary RPE cells. These results were also observed by confocal laser microscopy. These findings indicated that RPE metaplasia resulting in a change of biological cell behavior might be a necessary predisposing step in the development of PVR.

A New Family of Cdc42 Effector Proteins, CEPs, Function in Fibroblast and Epithelial Cell Shape Changes

Cdc42, a Rho GTPase, regulates the organization of the actin cytoskeleton by its interaction with several distinct families of downstream effector proteins. Here, we report the identification of four new Cdc42-binding proteins that, along with MSE55, constitute a new family of effector proteins. These molecules, designated CEPs, contain three regions of homology, including a Cdc42 binding domain and two unique domains called CI and CII. Experimentally, we have verified that CEP2 and CEP5 bind Cdc42. Expression of CEP2, CEP3, CEP4, and CEP5 in NIH-3T3 fibroblasts induced pseudopodia formation. Fibroblasts coexpressing dominant negative Cdc42 with CEP2 or expressing a Cdc42/Rac interactive binding domain mutant of CEP2 did not induce pseudopodia formation. In primary keratinocytes, CEP2- and CEP5-expressing cells showed reduced F-actin localization at the adherens junctions with an increase in thin stress fibers that extended the length of the cell body. Keratinocytes expressing CEPs also showed an altered vinculin distribution and a loss of E-cadherin from adherens junctions. Similar effects were observed in keratinocytes expressing constitutively active Cdc42, but were not seen with a Cdc42/Rac interactive binding domain mutant of CEP2. These results suggest that CEPs act downstream of Cdc42 to induce actin filament assembly leading to cell shape changes.

Distinct involvement of cdc42 and RhoA GTPases in actin organization and cell shape in untransformed and Dbl oncogene transformed NIH3T3 cells.

The Dbl oncogene is a putative exchange factor for the small GTPases RhoA and Cdc42, which are involved in actin polymerization into stress fibers and filopodia, respectively. We report here that, upon adhesion to fibronectin, Dbl-transformed NIH3T3 cells display a contracted, polygonal shape with a high number of short stress fibers. In contrast, untransformed NIH3T3 cells acquire the characteristic fibroblast morphology and organize a regular mesh of long stress fibers. We show that in Dbl-transformed and in untransformed NIH3T3 cells the different shape and actin cytoskeleton organization observed in the early steps of adhesion involves activation of distinct GTPases. Upon adhesion to fibronectin, cell morphology of Dbl-transformed NIH3T3 cells depends on activation of RhoA and not of Cdc42. In contrast Cdc42 activation is necessary to untransfected NIH3T3 cells to acquire their fibroblast shape. In both Dbl-transformed and in untransformed NIH3T3 cells a basal Rac activation is necessary to support stress fiber organization, while constitutive Rac activation promotes ruffles and lamellipodia formation. As a consequence of RhoA activation, Dbl-transformed cells show high activity of ROCK-alpha and CRIK kinases, two known RhoA effectors. In addition Dbl-transformed and NIH3T3 cells expressing the constitutive active form of RhoA are less motile on fibronectin than cells expressing constitutive active Cdc42. We conclude that in NIH3T3 cells in response to fibronectin the expression of the Dbl oncogene leads to a predominant activation of RhoA which both supports the peculiar cell shape and actin cytoskeleton organization in stress fibers and regulates cell motility.

The influence of the successive depolymerization and polymerization of cytoskeleton components on the fibroblast shapes.

Monitoring the influence of the cytoskeleton polymers on the shape of fibroblasts, performing the experiments of repeated degradation and polymerization of microtubules and microfilaments, we found out that the presence of microtubules is necessary in order to regenerate the proper functional structure of microfilaments, and vice versa.