We studied the effect of flurbiprofen, a non-steroidal anti-inflammatory drug, on muscles that had been subjected to exercise-induced injury. The muscles of the anterior compartment in the limbs of rabbits were cyclically activated as the ankle was simultaneously moved through passive plantar flexion every two seconds for thirty minutes. This treatment imposed acute passive lengthening (eccentric contractions) of the maximally contracted muscles of the anterior compartment. After the eccentric contraction-induced muscle injury, one group of rabbits was treated with oral administration of flurbiprofen, two times a day for six days, while the other group of rabbits served as untreated controls. The contractile, histological, and ultrastructural properties of the muscles were measured before the initial exercise and at three, seven, and twenty-eight days afterward. The group that was treated with flurbiprofen demonstrated a more complete functional recovery than the untreated controls at three and seven days but had a deficit in torque and force generation at twenty-eight days. The administration of flurbiprofen also resulted in a dramatic preservation of the intermediate filament protein desmin. After three days, the proportion of fibers of the extensor digitorum longus that lost desmin-staining was significantly greater in the untreated controls than in the treated animals (34 +/- 4.1 compared with 2.9 +/- 1.7 per cent) (p < 0.001), a finding that supports the concept of a short-term protective effect. However, the muscles in the treated animals still mounted a dramatic regenerative response, as indicated by the expression of embryonic myosin. Early in the recovery period (at three days), significantly fewer fibers of the extensor digitorum longus (2.2 +/- 1.4 per cent) expressed embryonic myosin in the treated animals than in the untreated controls (11.8 +/- 1.9 per cent) (p < 0.001). However, at seven days, the expression of embryonic myosin by the muscles from the treated animals (19.5 +/- 11.9 per cent) actually exceeded that of the muscles from the untreated controls (16.2 +/- 4.1 per cent). This finding suggests either a delayed or an ineffectual regenerative response by the muscles in the treated animals.

We present the structure and nucleotide sequence of a gene encoding the human epidermal 67-kDa keratin. Three genomic clones were isolated from a lambda Charon 4A human genomic library by hybridization to a specific cDNA probe. One clone of 12.3 kilobase pairs was shown by R-loop, DNA sequence, and primer-extension analyses to encode an entire gene of about 6.25 kilobase pairs. Of eight identified introns, seven are located within the region that encodes the central coiled-coil alpha-helical domain of the protein. Except for one intron located at the end of the region encoding this domain, these do not delineate apparent structural subdomains. The positions of five of the introns exactly coincide with the positions of introns previously reported in the hamster gene for the intermediate filament protein vimentin [Quax, W., Egberts, W.V., Hendricks, W., Quax-Jeuken, Y. & Bloemandal, H. (1983) Cell 35, 215-233]. These findings suggest that the human 67-kDa keratin and vimentin genes arose from a common ancestral gene.

The small guanosine triphosphatase Ran loaded with GTP (RanGTP) can stimulate assembly of the type V intermediate filament protein lamin B into a membranous lamin B spindle matrix, which is required for proper microtubule organization during spindle assembly. Microtubules in turn enhance assembly of the matrix. Here we report that the isolated matrix contains known spindle assembly factors such as dynein and Nudel. Using spindle assembly assays in Xenopus egg extracts, we show that Nudel regulates microtubule organization during spindle assembly independently of its function at kinetochores. Importantly, Nudel interacts directly with lamin B to facilitate the accumulation and assembly of lamin-B-containing matrix on microtubules in a dynein-dependent manner. Perturbing either Nudel or dynein inhibited the assembly of lamin B matrix. However, depleting lamin B still allowed the formation of matrices containing dynein and Nudel. Therefore, dynein and Nudel regulate assembly of the lamin B matrix. Interestingly, we found that whereas depleting lamin B resulted in disorganized spindle and spindle poles, disrupting the function of Nudel or dynein caused a complete lack of spindle pole focusing. We suggest that Nudel regulates microtubule organization in part by facilitating assembly of the lamin B spindle matrix in a dynein-dependent manner.

Treatment of isolated rat hepatocptes with low concentrations of digitonin increases the permeability of the plsma membrane to cytosolic proteins without causing release of organelles such as mitochondria into the surrounding medium. Electron microscopy showed that treatment of the cells with increasing concentations of digitonin results in a progressive loss in the continuity of the plasma membrane, while most other aspects of cellular morphology remain normal. Depletion of background staining material from the cytosol by digitonin treatment of the cells greatly enhances the visualization of the cytoskeleton. The use of this technique, together with immunofluorescent light microscopy, has verified the presence of an actin-containing filamentous network at the hepatocyte cortex as well as intermediate filaments distributed throughout the cell. Digitonin is thus useful both for selectively permeabilizing the plasma membrane and for intensifying the appearance of intracellular structures such as microfilaments that are normally difficult to observe in cells such as hepatocytes.

During the terminal stage of skeletal myogenesis, myoblasts stop replicating, fuse to form multinucleate fibers, and express the genes that encode the proteins that convey contractile capacity. Because of this dramatic shift in proliferative state, morphology, and gene expression, it has been possible to readily identify and quantitate terminally differentiating myoblasts. In contrast, it is not clear whether the proliferating cells that give rise to postmitotic myoblasts are equally distinct in their phenotype and in fact whether distinct stages in skeletal myogenesis precede the onset of terminal differentiation. To address these questions, monoclonal antibodies and immunofluorescence microscopy were used to determine that replicating myoblasts from newborn rats do express a muscle-specific phenotype. To identify replicating cells, incorporation of 5-bromo-2'-deoxyuridine (BrdUrd) into DNA was assayed by using anti-BrdUrd antibody. The developmentally regulated, muscle-specific, integral membrane protein H36 and the intermediate-filament protein desmin were scored as markers of the myogenic phenotype. The percentage of BrdUrd+ (i.e., proliferative) cells among H36+ and desmin+ myoblasts was equal to the percentage of BrdUrd+ cells in the entire population, indicating that the expression of H36 and desmin is uniformly characteristic of replicating myoblasts. Inhibition of protein synthesis before and during growth in BrdUrd did not alter the frequency of desmin and H36 immunofluorescence in BrdUrd+ cells. Thus, desmin and H36 were present in the replicating myoblasts prior to the onset of growth in BrdUrd. These results were confirmed using H36+ cells selected by flow cytometry: these purified H36+ myoblasts replicate, express desmin, and differentiate. Similar results were obtained with mouse myoblasts. Desmin expression in these mammalian cells differs from that in chicken embryo myoblasts: only a small proportion of replicating chicken embryo myoblasts express desmin. That replicating mammalian myoblasts have a muscle-specific phenotype serves to define a distinct stage in myogenic development and a specific cell in the myogenic lineage. Further, it implies that there is a regulatory event activated during myogenesis that precedes terminal differentiation and that is required for expression of those genes whose products distinguish the replicating myoblast.

We have established the complete coding sequence of the human vimentin gene. It had 91% homology to the coding sequence of the Syrian hamster vimentin gene (Quax et al., Cell 35:215-223, 1983) and partial homology to several other sequences coding for intermediate filament proteins. The most striking difference between the Syrian hamster and human vimentin genes was in the 3' untranslated region, which was considerably longer in the Syrian hamster. Using RNA blots and a human vimentin cDNA clone from an Okayama-Berg library, we have established that expression of the vimentin gene was growth regulated. The steady-state levels of cytoplasmic vimentin mRNA in 3T3 cells were increased by serum and platelet-derived growth factor, but not by epidermal growth factor, insulin, or platelet-poor plasma. The increase in expression of the vimentin gene that occurred when G0-phase cells were stimulated to proliferate was detected in six different cell types from four different species. The expression of the vimentin gene was also increased when HL60 cells were induced to differentiate by phorbol esters; it decreased when differentiation was induced by retinoic acid.

Synemin, a high-molecular-weight protein associated with intermediate filaments in muscle, and vimentin, an intermediate-filament subunit found in many different cell types, have been identified by immunologic and electrophoretic criteria as components of intermediate filaments in mature avian erythrocytes. Desmin, the predominant subunit of intermediate filaments in muscle, has not been detected in these cells. Two dimensional immunoautoradiography of proteolytic fragments of synemin and vimentin demonstates that the erythrocyte proteins are highly homologous, if not identical, to their muscle counterparts. Double immunoflurorescence reaveals that erythrocyte synemin and vimentin co-localize in a cytoplasmic network of sinuous filaments that extends from the nucleus to the plasma membrane and resists aggregation by colcemid. Erythrocytes that are attached to glass cover slips can be sonicated to remove nuclei and nonadherent regions of the plasma membrane; this leaves elliptical patches of adherent membrane that retain mats of vimentin- and synemin-containing intermediate filaments, as seen by immunofluorescence and rotary shadowing. Similarly, mechanical enucleation of erythrocyte ghosts in suspension allows isolation of plasma membranes that retain a significant fraction of the synemin and vimentin, as assayed by electrophoresis, and intermediate filaments, as seen in thin sections. Both synemin and vimentin remain insoluble along with spectrin and actin, in solutions containing nonionic detergent and high salt. However, brief exposure of isolated membrane to distilled water releases the synemin and vimentin together in nearly pure form, before the release of significant amounts of spectrin and actin. These data suggest that avian erythrocyte intermeditate filaments are somehow anchored to the plasma membrane; erythrocytes may thus provide a simple system for the study of intermediate filaments and their mode of interaction with membranes. In addition, these data, in conjunction with previous data from muscle, indicate that synemin is capable of associating with either desmin or vimentin and may thus perform a special role in the structure or function of intermediate filaments in erythrocytes as well as muscle.

Exposure of chicken cells grown in tissue culture to heat shock or sodium arsenite results in a dramatic increase in the synthesis of three major polypeptides with molecular weights of 83,000 (HSP 83), 68,000 (HSP 68; referred to here as "thermin"), and 25,000 (HSP 25). Incubation of BHK-21 or HeLa cells under the same conditions results in induction of HSP 68 and a 66,000-dalton polypeptide (HSP 66). Chicken thermin is resolved by isoelectric focusing into a major acidic and a more-basic component; mammalian thermin is resolved only into one major acidic component. HSP 83 and the acidic form of thermin are highly conserved in all avian and mammalian cells examined as judged by their electrophoretic mobilities, isoelectric points, and one-dimensional peptide maps. In addition, the acidic form of thermin is indistinguishable from a protein that copurifies with brain microtubules and that remains associated with the intermediate filament-enriched Triton/KCl cytoskeletons of cells grown in tissue culture. Thermin is also a component of skeletal myofibrils. HSP 83 and thermin are methylated in cells cultured under normal growth conditions. Induction of heat shock proteins by incubation of cells in the presence of sodium arsenite results in a marked methylation of the newly synthesized thermin. Under the same experimental conditions, no significant increase in methylation of the HSP 83 is observed. HSP 25 is not methylated in untreated cells or in cells treated with sodium arsenite. These results suggest that methylation of heat shock proteins may have an important role in regulating their function.

Using light and electron microscopic immunolocalization with antibodies to cytoskeletal proteins, we have characterized the nonlymphoid cells of various human lymphoid organs (lymph nodes, tonsils, spleen). In all these tissues, the lymphoid follicles contain a three-dimensional meshwork of "dendritic reticulum cells" which are characterized by the presence of desmosomal junctions, as demonstrated by positive punctate staining with antibodies to the desmosome-specific proteins desmoplakin I and desmoglein, and by intermediate-sized filaments (IFs) of the vimentin type only. In contrast, the extrafollicular regions are characterized by an extended meshwork of other types of reticulum cells, which also contain vimentin IFs but lack desmosomal proteins. In addition, a considerable, although variable proportion of these extrafollicular reticulum cells forms IFs containing cytokeratins 8 and 18 and/or desmin-containing IFs. The occurrence of cytokeratins 8 and 18 in lymph nodes has also been shown by gel electrophoresis and immunoblotting. Results of double-label immunolocalization indicate that some of the extrafollicular reticulum cells coexpress all three kinds of IF protein. A large proportion of these cells also synthesizes another marker of myogenic differentiation, i.e., the isoform of alpha-actin specific for smooth muscle. This proportion includes some cells that are negative for desmin. Comparison of the distribution of cells expressing cytokeratins and/or desmin with that of reticulum cells showing strong alkaline phosphatase activity (as a marker for the so-called "fiber-associated (fibroblastic) reticulum cells") suggests that the former represent a subset of the latter. The biological meaning of these different patterns of expression in reticulum cells and of the resulting cell-type heterogeneity as well as possible implications of these observations for tumor diagnosis, notably of lymph-node metastases and lymphomas, are discussed.

The localization of pp60src within adhesion structures of epithelioid rat kidney cells transformed by the Schmidt-Ruppin strain of Rous sarcoma virus was compared to the organization of actin, alpha-actinin, vinculin (a 130,000-dalton protein), tubulin, and the 58,000-dalton intermediate filament protein. The adhesion structures included both adhesion plaques and previously uncharacterized adhesive regions formed at cell-cell junctions. We have termed these latter structures "adhesion junctions." Both adhesion plaques and adhesion junctions were identified by interference-reflection microscopy and compared to the location of pp60src and the various cytoskeletal proteins by double fluorescence. The results demonstrated that the src gene product was found within both adhesion plaques and the adhesion junctions. In addition, actin, alpha-actinin, and vinculin were also localized within the same pp60src-containing adhesion structures. In contrast, tubulin and the 58,000-dalton intermediate filament protein were not associated with either adhesion plaques or adhesion junctions. Both adhesion plaques and adhesion junctions were isolated as substratum-bound structures and characterized by scanning electron microscopy. Immunofluorescence revealed that pp60src, actin, alpha-actinin, and vinculin were organized within specific regions of the adhesion junctions. Heavy accumulations of actin and alpha-actinin were found on both sides of the junctions with a narrow gap of unstained material at the midline, whereas pp60src stain was more intense in this central region. Antibody to vinculin stained double narrow lines defining the periphery of the junctional complexes but was excluded from the intervening region. In addition, the distribution of vinculin relative to pp60src within adhesion plaques suggested an inverse relationship between the presence of these two proteins. Overall, these results establish a close link between the src gene product and components of the cytoskeleton and implicate the adhesion plaques and adhesion junctions in the mechanism of Rous sarcoma virus-induced transformation.

Tumour-associated fibroblasts (TAFs) are part of the tumour stroma, providing functional and structural support for tumour progression and development. The origin and biology of TAFs are poorly understood, but within the tumour environment, TAFs become activated and secrete different paracrine and autocrine factors involved in tumorigenesis. It has been shown that bone marrow mesenchymal stem cells (MSCs) can be recruited into the tumours, where they proliferate and acquire a TAF-like phenotype. We attempted to determine to what extent TAFs characteristics in vitro juxtapose to MSCs' definition, and we showed that TAFs and MSCs share immunophenotypic similarities, including the presence of certain cell surface molecules [human leukocyte antigen-DR subregion (HLA-DR), CD29, CD44, CD73, CD90, CD106 and CD117]; the expression of cytoskeleton and extracellular matrix proteins, such as vimentin, α-smooth muscle actin, nestin and trilineage differentiation potential (to adipocytes, chondrocytes and osteoblasts). When compared to MSCs, production of cytokines, chemokines and growth factors showed a significant increase in TAFs for vascular endothelial growth factor, transforming growth factor-β1, interleukins (IL-4, IL-10) and tumour necrosis factor α. Proliferation rate was highly increased in TAFs and fibroblast cell lines used in our study, compared to MSCs, whereas ultrastructural details differentiated the two cell types by the presence of cytoplasmic elongations, lamellar content lysosomes and intermediate filaments. Our results provide supportive evidence to the fact that TAFs derive from MSCs and could be a subset of 'specialized' MSCs.

G protein-coupled receptor 30 [G protein-coupled estrogen receptor 1 (GPER1)], has been introduced as a membrane estrogen receptor and a candidate cancer biomarker and therapeutic target. However, several questions surround the subcellular localization and signaling of this receptor. In native cells, including mouse myoblast C(2)C(12) cells, Madin-Darby canine kidney epithelial cells, and human ductal breast epithelial tumor T47-D cells, G-1, a GPER1 agonist, and 17β-estradiol stimulated GPER1-dependent cAMP production, a defined plasma membrane (PM) event, and recruitment of β-arrestin2 to the PM. Staining of fixed and live cells showed that GPER1 was localized both in the PM and on intracellular structures. One such intracellular structure was identified as cytokeratin (CK) intermediate filaments, including those composed of CK7 and CK8, but apparently not endoplasmic reticulum, Golgi, or microtubules. Reciprocal coimmunoprecipitation of GPER1 and CKs confirmed an association of these proteins. Live staining also showed that the PM receptors constitutively internalize apparently to reach CK filaments. Receptor localization was supported using FLAG- and hemagglutinin-tagged GPER1. We conclude that GPER1-mediated stimulation of cAMP production and β-arrestin2 recruitment occur in the PM. Furthermore, the PM receptors constitutively internalize and localize intracellularly on CK. This is the first observation that a G protein-coupled receptor is capable of associating with intermediate filaments, which may be important for GPER1 regulation in epithelial cells and the relationship of this receptor to cancer.

The intermediate filament protein desmin is an essential component of the extra-sarcomeric cytoskeleton in muscle cells. This three-dimensional filamentous framework exerts central roles in the structural and functional alignment and anchorage of myofibrils, the positioning of cell organelles and signaling events. Mutations of the human desmin gene on chromosome 2q35 cause autosomal dominant, autosomal recessive, and sporadic myopathies and/or cardiomyopathies with marked phenotypic variability. The disease onset ranges from childhood to late adulthood. The clinical course is progressive and no specific treatment is currently available for this severely disabling disease. The muscle pathology is characterized by desmin-positive protein aggregates and degenerative changes of the myofibrillar apparatus. The molecular pathophysiology of desminopathies is a complex, multilevel issue. In addition to direct effects on the formation and maintenance of the extra-sarcomeric intermediate filament network, mutant desmin affects essential protein interactions, cell signaling cascades, mitochondrial functions, and protein quality control mechanisms. This review summarizes the currently available data on the epidemiology, clinical phenotypes, myopathology, and genetics of desminopathies. In addition, this work provides an overview on the expression, filament formation processes, biomechanical properties, post-translational modifications, interaction partners, subcellular localization, and functions of wild-type and mutant desmin as well as desmin-related cell and animal models.

Frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U) is the most common neuropathology associated with the clinical syndrome of frontotemporal dementia (FTD). Recently, TDP-43 was identified as the ubiquitinated pathological protein in both FTLD-U and sporadic amyotrophic lateral sclerosis. Although a number of studies have now confirmed that most sporadic and familial cases of FTLD-U are TDP-43 proteinopathies, there are exceptions. We describe six cases of early onset FTD with FTLD-U pathology that was negative for TDP-43, which we refer to as 'atypical' FTLD-U. All cases were sporadic and had very early onset FTD (mean age = 35 years), characterized by severe progressive psychobehavioural abnormalities in the absence of significant aphasia, cognitive-intellectual dysfunction or motor features. The neuropathological features were highly consistent, with small, round, neuronal cytoplasmic inclusions that were immunoreactive for ubiquitin (ub-ir), but negative for tau, alpha-synuclein, intermediate filaments and TDP-43. Cytoplasmic inclusions were most numerous in the neocortex, dentate granule cells and hippocampal pyramidal neurons. Ub-ir neuronal intra-nuclear inclusions were also present in neocortical and hippocampal neurons and had the unusual appearance of straight, curved or twisted filaments. We believe that these cases represent a new entity that is clinically and pathologically distinct from all currently recognized subtypes of FTLD. Moreover, the existence of such cases indicates that the designations of 'FTLD-U' and 'TDP-43 proteinopathy' should not be considered to be synonymous.

The activation of N-methyl-D-aspartate-receptors (NMDARs) in synapses provides plasticity and cell survival signals, whereas NMDARs residing in the neuronal membrane outside synapses trigger neurodegeneration. At present, it is unclear how these opposing signals are transduced to and discriminated by the nucleus. In this study, we demonstrate that Jacob is a protein messenger that encodes the origin of synaptic versus extrasynaptic NMDAR signals and delivers them to the nucleus. Exclusively synaptic, but not extrasynaptic, NMDAR activation induces phosphorylation of Jacob at serine-180 by ERK1/2. Long-distance trafficking of Jacob from synaptic, but not extrasynaptic, sites depends on ERK activity, and association with fragments of the intermediate filament α-internexin hinders dephosphorylation of the Jacob/ERK complex during nuclear transit. In the nucleus, the phosphorylation state of Jacob determines whether it induces cell death or promotes cell survival and enhances synaptic plasticity.

Keratins make up the largest subgroup of intermediate filament (IF) proteins and form a dynamic network of 10-12 nm filaments, built from type I/type II heterodimers, in the cytoplasm of epithelial cells. A major function of keratin IFs is to protect epithelial cells from mechanical and non-mechanical stresses that cause cell rupture and death. Interference with this role is the root cause of a large number of inherited epithelial fragility conditions. Additional functions, non-mechanical in nature, are manifested in a way that depends on the specific keratin and on the epithelial context. The recent discovery of unusual mutations affecting keratin proteins has uncovered a novel dimension of their mechanical support function, and has synergized with mouse genetics to reveal a role in skin pigmentation. Other studies extended the role of keratin proteins in regulating the response to pro-apoptotic signals, and revealed their ability to modulate protein synthesis and cell size in epithelial cells challenged to grow.

Intermediate filaments (IFs) are a major component of the cytoskeleton in astrocytes. Their role is far from being completely understood. Immature astrocytes play a major role in neuronal migration and neuritogenesis, and their IFs are mainly composed of vimentin. In mature differentiated astrocytes, vimentin is replaced by the IF protein glial fibrillary acidic protein (GFAP). In response to injury of the CNS in the adult, astrocytes become reactive, upregulate the expression of GFAP, and reexpress vimentin. These modifications contribute to the formation of a glial scar that is obstructive to axonal regeneration. Nevertheless, astrocytes in vitro are considered to be the ideal substratum for the growth of embryonic CNS axons. In the present study, we have examined the potential role of these two major IF proteins in both neuronal survival and neurite growth. For this purpose, we cocultured wild-type neurons on astrocytes from three types of knock-out (KO) mice for GFAP or/and vimentin in a neuron-astrocyte coculture model. We show that the double KO astrocytes present many features of immaturity and greatly improve survival and neurite growth of cocultured neurons by increasing cell-cell contact and secreting diffusible factors. Moreover, our data suggest that the absence of vimentin is not a key element in the permissivity of the mutant astrocytes. Finally, we show that only the absence of GFAP is associated with an increased expression of some extracellular matrix and adhesion molecules. To conclude, our results suggest that GFAP expression is able to modulate key biochemical properties of astrocytes that are implicated in their permissivity.

The differentiation of the predominant cell types of the mucosal epithelium of the mammalian gastrointestinal tract is characterized by increasing amounts of an intermediate-sized filament (IF) protein designated cytokeratin (CK) 20 which is a major cellular protein of mature enterocytes and goblet cells. Here we report the isolation of the human gene encoding CK 20, its complete nucleotide sequence and the amino acid sequence deduced therefrom that identifies this polypeptide (mol. wt. 48553) as a member of the type I-CK subfamily. Remarkable, however, is the comparably great sequence divergence of CK 20 from all other known type I-CKs, with only 58% identical amino acids in the conserved alpha-helical 'rod' domain of CK 20 and, e.g. CK 14. Using riboprobes corresponding to exon 6 of the gene in Northern blot and ribonuclease protection assays, we show that the approximately 1.75 kb mRNA encoding CK 20 is specifically produced in cells of the intestinal and gastric mucosa, including tumors and cell lines derived therefrom. The appearance of CK 20-positive cells in human embryonic and fetal development and in adult tissues has been studied using immunohistochemistry with CK 20-specific antibodies. CK 20 synthesis has first been recognized at embryonic week 8 in individual 'converted' simple epithelial cells of the developing intestinal mucosa. In later fetal stages, CK 20 synthesis extends over most goblet cells and a variable number of villus enterocytes. The distribution of CK 20-positive cells in the developing gastric and intestinal mucosa is similar to--but not identical with--the pattern in the adult intestine in which all enterocytes and goblet cells as well as certain 'low-differentiated' columnar cells contain CK 20, whereas the neuroendocrine ('enterochromaffin') and Paneth cells are negative. In gastrointestinal carcinomas similarly examined, CK 20 has been detected in almost all cases (50/52) of colorectal adenocarcinomas, including all grades of differentiation and malignancy and also metastatic tumors, whereas CK 20 immunostaining in gastric carcinomas has been found less consistent and more heterogeneous. The possible biological meaning of the specific expression of the CK 20 gene in certain cells of the gastrointestinal tract and carcinomas derived therefrom and the regulatory mechanisms involved in the integration of the protein in the IF cytoskeleton are discussed.

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.

Keratins are cytoplasmic intermediate filament proteins preferentially expressed by epithelial tissues in a site-specific and differentiation-dependent manner. The complex network of keratin filaments in stratified epithelia is tightly regulated during squamous cell differentiation. Keratin 14 (K14) is expressed in mitotically active basal layer cells, along with its partner keratin 5 (K5), and their expression is down-regulated as cells differentiate. Apart from the cytoprotective functions of K14, very little is known about K14 regulatory functions, since the K14 knockout mice show postnatal lethality. In this study, K14 expression was inhibited using RNA interference in cell lines derived from stratified epithelia to study the K14 functions in epithelial homeostasis. The K14 knockdown clones demonstrated substantial decreases in the levels of the K14 partner K5. These cells showed reduction in cell proliferation and delay in cell cycle progression, along with decreased phosphorylated Akt levels. K14 knockdown cells also exhibited enhanced levels of activated Notch1, involucrin, and K1. In addition, K14 knockdown AW13516 cells showed significant reduction in tumorigenicity. Our results suggest that K5 and K14 may have a role in maintenance of cell proliferation potential in the basal layer of stratified epithelia, modulating phosphatidylinositol 3-kinase/Akt-mediated cell proliferation and/or Notch1-dependent cell differentiation.

The intermediate filament proteins, A- and B-type lamins, form the nuclear lamina scaffold adjacent to the inner nuclear membrane. B-type lamins confer elasticity, while A-type lamins lend viscosity and stiffness to nuclei. Lamins also contribute to chromatin regulation and various signaling pathways affecting gene expression. The mechanical roles of lamins and their functions in gene regulation are often viewed as independent activities, but recent findings suggest a highly cross-linked and interdependent regulation of these different functions, particularly in mechanosignaling. In this newly emerging concept, lamins act as a "mechanostat" that senses forces from outside and responds to tension by reinforcing the cytoskeleton and the extracellular matrix. A-type lamins, emerin, and the linker of the nucleoskeleton and cytoskeleton (LINC) complex directly transmit forces from the extracellular matrix into the nucleus. These mechanical forces lead to changes in the molecular structure, modification, and assembly state of A-type lamins. This in turn activates a tension-induced "inside-out signaling" through which the nucleus feeds back to the cytoskeleton and the extracellular matrix to balance outside and inside forces. These functions regulate differentiation and may be impaired in lamin-linked diseases, leading to cellular phenotypes, particularly in mechanical load-bearing tissues.

Evidence from a variety of sources suggests that pericytes have contractile properties and may therefore function in the regulation of capillary blood flow. However, it has been suggested that contractility is not a ubiquitous function of pericytes, and that pericytes surrounding true capillaries apparently lack the machinery for contraction. The present study used a variety of techniques to investigate the expression of contractile proteins in the pericytes of the CNS. The results of immunocytochemistry on cryosections of brain and retina, retinal whole-mounts and immunoblotting of isolated brain capillaries indicate strong expression of the smooth muscle isoform of actin (alpha-SM actin) in a significant number of mid-capillary pericytes. Immunogold labelling at the ultrastructural level showed that alpha-SM actin expression in capillaries was exclusive to pericytes, and endothelial cells were negative. Compared to alpha-SM actin, non-muscle myosin was present in lower concentrations. By contrast, smooth muscle myosin isoforms, were absent. Pericytes were strongly positive for the intermediate filament protein vimentin, but lacked desmin which was consistently found in vascular smooth muscle cells. These results add support for a contractile role in pericytes of the CNS microvasculature, similar to that of vascular smooth muscle cells.

A hundred years after the first description, many aspects of pericytes remain to be examined. Mesenchymal in origin, pericytes form an incomplete envelopment around the endothelial cells and within the microvascular basement membrane of capillaries and postcapillary venules. Morphologically, they appear as long, slender, polymorphic cells, showing an elongated cell body, from which arise longitudinal and circumferential branches. Cell bodies and cytoplasmic processes of pericytes, as well as the endothelial cells, are enveloped by the same basal lamina, except for where they make direct contacts with each other. The pericyte/endothelial cell contacts are peg and socket, adhesion plaques and gap junctions, making up structural mechanisms for force transmission and a possible receptor system for cells, in which the pericyte and endothelial cells respond to secondary signals generated in the other cells. Electron microscopic studies have revealed an elaborate network of cytoplasmic filaments. Pericyte intermediate filament proteins show species and tissue differences, expressing vimentin or vimentin and desmin. The pericytes also express protein typical of contractile cells, i.e. smooth muscle-specific isoforms of actin and myosin, cyclic GMP-protein kinase and tropomyosin. A gradual transition is observed between pericytes and smooth muscle cells in both terminal arterioles and venules. Several general functions for the pericytes have been postulated: contractability; permeability regulator; integrity maintainer; endothelial cell growth modulator; and cell progenitor with considerable mesenchymal potential.

Recombinant plasmids were made containing cDNAs synthesized on hamster mRNAs coding for cytoskeletal (beta- or gamma-) actins and for vimentin. Hybridization of the actin probe on restriction digests of one avian and five mammalian DNAs yielded multiple bands; the vimentin probe revealed only one band (accompanied by 2-3 faint bands in some DNAs). The results obtained with the vimentin probe indicate that the corresponding coding sequences: (a) are highly conserved in warm-blooded vertebrates like the actin sequences; (b) have strongly diverged from those coding for other intermediate filament proteins, since hybridization of the vimentin probe does not lead to a diagnostic multiband pattern; and (c) most likely contribute to single gene, in contrast to the sequences coding for other cytoskeletal proteins. Hybridization of the probes on mRNAs from the different sources used showed that the non-coding sequences of both vimentin and actin genes are conserved in length.