Intermediate filaments and cytoplasmic networking: new connections and more functions.

Recent research highlights the roles of cytoskeletal intermediate filaments (IFs) and their interactions with both the cell surface and other cytoskeletal systems in maintaining cellular integrity and the mechanical properties of cytoplasm. This has been demonstrated by analyses of mutations in IF-associated proteins (IFAPs) that are involved in connecting IFs to cell surface junctions. New data also point to the role of IFAPs as molecular 'nuts and bolts' in the construction of an integrated cytoplasmic architecture. This is highlighted by the initial descriptions of a family of multifunctional molecules that are capable of bridging IFs to other cytoskeletal elements. These findings, together with the development of specific peptide inhibitors capable of disassembling IF networks in vivo, are paving the way to the identification of new cellular functions for IFs and IFAPs.

Intermediate filaments: not so tough after all.

The dynamic properties of cellular protein polymers such as microtubules and microfilaments depend to a large extent on the cell's capacity to modify rapidly the exchange rate between polymerized and unpolymerized pools of subunits. Until quite recently the dynamic nature of intermediate filaments was underestimated because of their biochemical stability in vitro and a paucity of studies on their characteristics in vivo. However, the recent studies described in this review show that the karyoskeletal and cytoskeletal structures that assemble from many intermediate filament proteins possess the properties expected of dynamic protein polymer networks.

IFAP 300 is common to desmosomes and hemidesmosomes and is a possible linker of intermediate filaments to these junctions.

The distribution of IFAP 300, a protein previously characterized as cross-linking vimentin intermediate filaments (IF), has been investigated in epithelial cells. In frozen sections of bovine tongue epithelium the staining obtained with IFAP 300 antibodies is concentrated in the peripheral cytoplasm of keratinocytes, including the entire peripheral region of basal cells. Further immunofluorescence studies reveal that in primary cultures of mouse keratinocytes the distribution of IFAP 300 is similar to that of the desmosomal protein desmoplakin. In rat bladder carcinoma 804G cells the staining pattern of IFAP 300 antibodies coincides with that obtained with antibodies against the hemidesmosomal protein BP 230. By immunogold electron microscopy IFAP 300 is mainly located at sites where IF appear to attach to desmosomes and hemidesmosomes. Morphometric analyses of the distribution of the gold particles show that IFAP 300 overlaps with desmoplakin and BP 230, but also that it extends deeper into the cytoplasm than these latter two proteins. The staining reaction seen in epithelial cells by immunofluorescence and immunogold is specific for IFAP 300 as shown by immunoblotting. Immunoblotting also reveals that IFAP 300 is present in both cell-free preparations of desmosomes and hemidesmosomes. These morphological and biochemical results are intriguing since, in recent years, the proteins appearing in these two types of junctions have been found to be different. One possible exception is plectin, a protein that has been suggested to be very similar to IFAP 300. However, we show here that IFAP 300 differs from plectin in several respects, including differences at the primary sequence level. We also show that purified IFAP 300 pellets with in vitro polymerized IF prepared from desmosome-associated keratins under conditions in which IFAP 300 alone is not sedimentable. This indicates that IFAP 300 can associate with keratin IF. These data, taken together with the immunogold results, suggest that IFAP 300 functions in epithelial cells as a linker protein connecting IF to desmosomes as well as to hemidesmosomes, possibly through structurally related proteins such as desmoplakin and BP 230, respectively.

A monoclonal antibody against alpha-smooth muscle actin: a new probe for smooth muscle differentiation.

A monoclonal antibody (anti-alpha sm-1) recognizing exclusively alpha-smooth muscle actin was selected and characterized after immunization of BALB/c mice with the NH2-terminal synthetic decapeptide of alpha-smooth muscle actin coupled to keyhole limpet hemocyanin. Anti-alpha sm-1 helped in distinguishing smooth muscle cells from fibroblasts in mixed cultures such as rat dermal fibroblasts and chicken embryo fibroblasts. In the aortic media, it recognized a hitherto unknown population of cells negative for alpha-smooth muscle actin and for desmin. In 5-d-old rats, this population is about half of the medial cells and becomes only 8 +/- 5% in 6-wk-old animals. In cultures of rat aortic media SMCs, there is a progressive increase of this cell population together with a progressive decrease in the number of alpha-smooth muscle actin-containing stress fibers per cell. Double immunofluorescent studies carried out with anti-alpha sm-1 and anti-desmin antibodies in several organs revealed a heterogeneity of stromal cells. Desmin-negative, alpha-smooth muscle actin-positive cells were found in the rat intestinal muscularis mucosae and in the dermis around hair follicles. Moreover, desmin-positive, alpha-smooth muscle actin-negative cells were identified in the intestinal submucosa, rat testis interstitium, and uterine stroma. alpha-Smooth muscle actin was also found in myoepithelial cells of mammary and salivary glands, which are known to express cytokeratins. Finally, alpha-smooth muscle actin is present in stromal cells of mammary carcinomas, previously considered fibroblastic in nature. Thus, anti-alpha sm-1 antibody appears to be a powerful probe in the study of smooth muscle differentiation in normal and pathological conditions.

Expression of class II transplantation antigen on vascular smooth muscle cells in human atherosclerosis.

A large proportion of the cells of the human atherosclerotic plaque is assumed to be derived from medial smooth muscle cells. In contrast to these, the cells of the plaque have the capacity to accumulate lipid, and they also proliferate at a higher rate than medial cells. It has therefore been suggested that smooth muscle cells undergo a change of phenotype during atherogenesis, but there has been no evidence for such a change on the molecular level. We have now analyzed carotid artery plaques using a battery of antibodies against cell surface and cytoskeletal antigens, and found that most of the cells express the class II transplantation antigen (Ia antigen) HLA-DR. Also, the beta chain of HLA-DR was detected by immunoblotting of plaque extracts with the OKIa1 monoclonal antibody. HLA-DR is normally present on cells of the immune system, but only 60% of the DR-positive cells of the plaque reacted with monoclonal antibodies specific for macrophages and lymphocytes. Many of the remaining DR-positive cells contained the muscle-specific intermediate filament protein, desmin. This indicates that smooth muscle cells of atherosclerotic plaques express DR antigen. In contrast, very few DR-positive cells were found in normal human arteries. This suggests that expression of class II antigen is part of a phenotypic change in smooth muscle cells in atherosclerosis.

The intermediate filament proteins of rabbit normal epidermal Merkel cells are cytokeratins.

Four hundred Merkel cells (MC) have been studied by double-label immunofluorescence using: (1) a monoclonal antibody which has been previously demonstrated to react with MC and (2) antisera and monoclonal antibodies against the 5 types of intermediate filaments. It was demonstrated that MC did not react with vimentin, desmin, glial acidic fibrillary protein, or neurofilament antisera. A strong staining of MC was observed with 2 antisera and 2 monoclonal antibodies against keratin. The cytokeratin polypeptide pattern of MC is probably similar to that of simple epithelia. These findings attest to the epithelial nature of MC.

Specific demonstration of myoepithelial cells by anti-alpha smooth muscle actin antibody.

The myoepithelial cells of the sweat, mammary, tracheobronchial, and salivary glands are specifically identified by monoclonal antibody alpha-SM-1, which recognizes alpha smooth muscle actin and not the other actin isoforms. Basal or "reserve" cells in the stratified epithelia and excretory ducts of the salivary glands are negative, as well as all other epithelial cells in various organs. The reaction can be performed in routinely fixed and embedded tissues and is of practical interest in diagnostic histopathology. In immunoelectron cytochemistry, alpha-SM-1 antibody binds to the microfilament bundles in myoepithelial cells of the breast, but does not stain luminal cells and occasional basally located epithelial cells. These basal cells are morphologically and immunocytochemically distinct from the myoepithelial cells, and their nature and significance remain to be clarified.

Alpha-smooth muscle actin, a differentiation marker of smooth muscle cells, is present in microfilamentous bundles of pericytes.

alpha-Smooth muscle (alpha-sm) actin, an isoform typical of smooth muscle cells (SMC) and present in high amounts in vascular SMC, was demonstrated in the cytoplasm of pericytes of various rat and human organs by means of immunocytochemistry at the electron microscopic level. In SMC and pericytes, alpha-sm actin was localized in microfilament bundles, strengthening the assumption that it is the functional isoform in these cell types and supporting the assumption that pericytes exert contractile functions.

Autoimmune syndrome after neonatal induction of tolerance to alloantigens: analysis of the specificity and of the cellular and genetic origin of autoantibodies.

BALB/c mice neonatally injected with 10(8) semiallogeneic (C57BL/6 x BALB/c)F1 spleen cells become tolerant to the H-2b alloantigens, but also develop a wide range of autoimmune manifestations characteristic of systemic lupus erythematosus (SLE). Indeed, in these mice, the presence of a hypergammaglobulinaemia, autoantibodies--including anti-ssDNA, anti-platelet, thymocytotoxic and rheumatoid factor antibodies--circulating immune complexes, cryoglobulins as well as renal glomerular deposition of immunoglobulins have been observed. In this study, we have shown that the allogenic effect and B cell chimaerism which characterize these F1 cell-injected mice is associated with the expression of a large spectrum of autoantibodies, including anti-ssDNA and anti-cytoskeleton antibodies, and that these autoantibodies are not multispecific. We took advantage of the fact that, in this model, autoantibodies are exclusively produced by F1 donor B cells to inject newborn BALB/c mice with F1 Xid spleen cells lacking the CD5+ B cell subset. Injection of 2 x 10(8) F1 Xid spleen cells triggers the production of anti-ssDNA as well as anti-BrMRBC antibodies, and these mice developed tissue lesions. Finally, analysis of the VH gene family expressed by monoclonal autoantibodies derived from F1 cell-injected mice showed that they used the 2 largest families J558 and 7183. These results suggest that the allogenic effect and B cell chimerism which characterize the neonatal induction of tolerance to MHC alloantigens is associated with the selective triggering of autoreactive B cells producing monospecific IgG autoantibodies. They also imply that upon stimulation by persisting alloreactive CD4+ T cells, either CD5- B cells are able to produce autoantibodies or autoantibody-producing CD5+ B cells can differentiate from Xid spleen cells.

Keratin expression in astrocytomas: an immunofluorescent and biochemical reassessment.

Several studies have shown that immunoenzymatic staining of formalin-fixed, paraffin-embedded astrocytomas with keratin antibodies frequently yields positive labelling, but no biochemical evidence of keratin expression in astrocytomas has been reported. We have investigated the presence of keratin in astrocytoma and normal brain tissues both by immunofluorescence on frozen sections and by 1D and 2D immunoblotting using seven monoclonal antibodies that, collectively, recognize most keratin polypeptides. Four of these antibodies did not stain neural tissues by immunofluorescence and were also negative by immunoblotting. The remaining three keratin antibodies stained normal brain and/or a high proportion of astrocytomas. Two of these three antibodies only stained glial fibrillary acidic protein (GFAP)-positive cells, while the third only stained GFAP-negative cells. 1D and 2D immunoblotting analysis showed that positive immunofluorescence staining of normal brain and/or astrocytomas seen with these three keratin antibodies was due to cross-reactivity with non-keratin proteins, such as GFAP. These results demonstrate that, contrary to earlier suggestions, keratin polypeptides are not frequently expressed in astrocytomas. Our studies also emphasize that keratin antibodies should be used cautiously for the differential diagnosis of undifferentiated gliomas from tumours of non-glial origin.

TGF-alpha induces a stationary, radial-glia like phenotype in cultured astrocytes.

Transgenic mice studies have suggested that transforming growth factor-alpha (TGF-alpha) influences the postnatal differentiation of astrocytes. To understand the role of TGF-alpha during astrocytic differentiation, it is important to determine how this factor affects astrocytes in the absence of other influences. We have thus examined in vitro under serum-free medium conditions the effect of TGF-alpha on the properties of astrocytes derived from the cerebral cortex of newborn rats. When TGF-alpha is added to serum-free medium, most astrocytes lose their polygonal shape and extend two long processes running in opposite directions. This bipolar morphology strikingly resembles that of radial glial cells. Intriguingly, serum inhibits this morphological transformation. TGF-alpha also triggers an increase in glial fibrillary acidic protein (GFAP) expression and a decrease in nestin expression. Another major effect of TGF-alpha is to practically abolish the motility of astrocytes. TGF-alpha, however, does not appear to influence the proliferation and apoptosis of astrocytes. These results suggest that polygonal astrocytes are derived primarily from radial glial cells, and that in vivo TGF-alpha may be instrumental in determining the shape and migratory potential of radial glial cells.

Remodeling of the aortic smooth muscle cell cytoskeleton during developmental and pathological conditions.

The remodeling of aortic smooth muscle cell cytoskeleton has been investigated qualitatively and quantitatively during rat aorta development and experimental or human atheromatosis, using immunofluorescent and biochemical techniques. The cytoskeleton of smooth muscle cells in the intimal thickening 15 days after endothelial removal and in human atheromatous plaque is very similar to that of poorly differentiated aortic smooth muscle cells of foetal and newborn rats. Our studies suggest that cytoskeletal changes (a switch in the synthesis of actin isoforms in particular) are reliable markers of proliferative aortic smooth muscle cells, and of atheromatous smooth muscle cells.

Expression of actin isoforms and intermediate filament proteins in childhood orbital rhabdomyosarcomas.

The diagnosis of orbital rhabdomyosarcoma (RMS) in childhood gives rise to several clinical and anatomo-pathological problems. Antibodies recognizing structural proteins and cytoskeletal components have been shown to increase the diagnostic accuracy of different neoplastic lesions. In this study we examined anatomo-clinically and, where possible, by means of immunohistochemistry and electron microscopy, a series of 14 cases of orbital RMS in childhood. In the 12 cases studied by immunohistochemistry, desmin was always present, although showing variable patterns, and alpha-sarcomeric actin was found in 10 cases. alpha-Smooth muscle actin was always absent. The other markers tested (myoglobin, polyclonal actin, vimentin and enolase) proved unreliable for several reasons. We conclude that antibodies against desmin and alpha-sarcomeric actin are useful for the diagnostic definition of RMS. In addition, immunohistochemical analysis supplies data regarding the degree of tumor differentiation and may be applied to monitor radio- and chemotherapy.

Identification of cadherin-11 down-regulation as a common response of astrocytoma cells to transforming growth factor-alpha.

Transforming growth factor-alpha (TGF-alpha) and its receptor are frequently co-expressed in high-grade astrocytomas, suggesting a role for TGF-alpha autocrine/paracrine loops in the malignant progression of astrocytomas. To identify genes that may be critical in mediating TGF-alpha impact on the malignant progression of astrocytomas, we have used cDNA arrays to investigate TGF-alpha effects on the gene expression profile of U-373 MG glioblastoma cells. We found that in these cells approximately 50% of the TGF-alpha regulated genes code for cell motility/invasion-related proteins. TGF-alpha action on the expression of four of these proteins, alpha-catenin, IQGAP1, RhoA, and cadherin-11, was further investigated by immunoblotting in four astrocytoma cell lines and in normal astrocytes. The results demonstrate that the effects of TGF-alpha on IQGAP1, alpha-catenin, and RhoA expression are cell-line dependent. On the other hand, under TGF-alpha treatment, cadherin-11 expression is consistently decreased in all astrocytoma cell lines tested but is increased in normal astrocytes. In addition, we found that cadherin-11 is consistently down-regulated in astrocytomas versus normal brain tissues. Altogether, these results suggest that the down-regulation of cadherin-11 is a frequent molecular event in the neoplastic transformation of astrocytes and that this down-regulation may be initiated and/or amplified by TGF-alpha autocrine/paracrine loops during tumor progression.

TGF-alpha differentially regulates GFAP, vimentin, and nestin gene expression in U-373 MG glioblastoma cells: correlation with cell shape and motility.

To begin understanding the regulation and biological significance of changes in the expression of intermediate filament proteins in astrocytic tumors, we have recently shown that TGF-alpha alters the protein level of glial fibrillary acidic protein (GFAP), nestin, and vimentin in U-373 MG glioblastoma cells. Here, we have determined the molecular mechanisms regulating these changes. In addition, to evaluate the significance of these changes we have examined whether TGF-alpha affects various cellular properties related to differentiation. Our results show that, in U-373 MG cells treated with TGF-alpha, GFAP gene transcription, mRNA level, and specific protein synthesis decrease by approximately 50%. This suggests that, in U-373 MG cells, TGF-alpha down-regulates the expression of this marker of astrocytic differentiation at the transcriptional level, resulting in decreased GFAP mRNA level and specific protein synthesis. In contrast, TGF-alpha does not change vimentin gene transcription, but increases by about 50% the transcription of the gene for nestin, a marker for undifferentiated astrocytic precursors. This differential regulation of GFAP, nestin, and vimentin gene expression indicates that TGF-alpha induces further dedifferentiation of U-373 MG cells. This notion is also supported by our findings that TGF-alpha increases the motility of U-373 MG cells and induces a less stellate morphology.

Actin-isoform pattern as a marker of normal or pathological smooth-muscle and fibroblastic tissues.

The relative proportions of actin isoforms present in smooth-muscle (SM) and fibroblastic human and non-human tissue extracts were examined by densitometric evaluation of Coomassie-Blue-stained spots in two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) as well as by quantification of radiolabeled actin NH2-terminal peptide spots separated by two-dimensional paper electrophoresis. SM tissues contained alpha- and gamma-SM as well as beta- and gamma-cytoplasmic (CY) actins in different proportions in different organs. Species differences with respect to the ratios of the isoactins were also observed. Moreover, during pregnancy, both human and rat myometrium exhibited a changed actin-isoform pattern, there being an increased proportion of gamma-actin. Analysis of the NH2-terminal peptides showed that, in human myometrium, this was essentially due to an increase in the amount of the gamma-SM isoform. Fibroblastic tissues were found to contain only the beta- and gamma-CY isoforms, the ratio being approximately 2.6:1. Thus, the presence or absence of alpha-actin provides a reliable biochemical criterion for distinguishing between fibroblastic and SM cell populations and/or tissues. This distinction and the evaluation of changes in isoactin ratios may be useful in the study of differentiation as well as physiological and pathological phenomena, and for determining the origin of certain soft-tissue tumours.

Correlation between the distribution of smooth muscle or non muscle myosins and alpha-smooth muscle actin in normal and pathological soft tissues.

The distribution of smooth muscle (SM) and non muscle myosins was compared with that of alpha-SM actin in various normal and pathological tissues and in cultured cells by means of indirect immunofluorescence using a monoclonal antibody specific for alpha-SM actin [anti-alpha sm-1, Skalli et al., 1986b] and two polyclonal antibodies raised against bovine aortic myosin (ABAM) and human platelet myosin (AHPM), respectively. In normal tissues ABAM stained vascular and parenchymal smooth muscle cells (SMC), myoepithelial cells and myoid cells of the testis in a pattern similar to that reported by other authors with antisera raised against non vascular SM myosin. Cells stained with ABAM were always positive for anti-alpha sm-1. In human and experimental atheromatous plaques, most cells were positive for AHPM; a variable proportion was also stained for ABAM plus anti-alpha sm-1. Myofibroblasts from rat granulation tissue, Dupuytren's nodule and stroma from breast carcinoma were constantly positive for AHPM and negative for ABAM; however, myofibroblasts from Dupuytren's nodule and breast carcinoma were anti-alpha sm-1 positive. Early primary cultures of rat aortic SMC were positive for ABAM and anti-alpha sm-1 and became negative for ABAM and positive for AHPM after a few days in culture. They remained positive for AHPM and anti-alpha sm-1 after passages; the staining of AHPM and anti-alpha sm-1 appeared to be colocalized along the same stress fibers. These results may be relevant for the understanding of SMC function and adaptation, and show that in non malignant SMC proliferation, alpha-SM actin represents a more general marker of SM origin than SM myosin.

Distribution of intermediate filament proteins in normal and diseased human glomeruli.

The distribution of intermediate filament proteins (vimentin, desmin, and cytokeratin) was studied by means of immunofluorescence in the normal human and rat glomerulus and in pathologic human glomeruli. Antifibronectin antibodies were used as mesangial markers. In normal human glomeruli, vimentin antibodies stained endothelial cells, podocytes, and mesangial cells; desmin antibodies, surprisingly, stained podocytes. In normal rat glomeruli, the pattern of vimentin staining was the same as in humans, but desmin antibodies stained both mesangial cells and podocytes. In human and rat glomeruli cytokeratin staining was confined to segments of Bowman's capsule. In human pathologic glomeruli, vimentin and desmin antibodies stained the structures that were positive in normal glomeruli, giving a characteristic pattern for each pathologic condition examined. These results are compatible with the mesenchymal origin of podocytes and mesangial cells and suggest that both cells have smooth muscle-like phenotypic features. Mesangial cells may have slightly different differentiation paths in humans and rats, leading to a distinct expression of intermediate filament proteins.

Alpha-smooth muscle actin is transiently expressed by myofibroblasts during experimental wound healing.

We have studied the expression of alpha-smooth muscle actin, smooth muscle myosin, and desmin in granulation tissue during the healing of an open wound in the rat by means of electron microscopy and immunohistochemistry, at the light and electron microscopic levels, using specific antibodies directed against these proteins. Important amounts of the three antigens were always expressed in pericytic and/or smooth muscle cells of neoformed small vessels. In fibroblastic cells, microfilaments were absent 4 days after wounding but accumulated gradually, starting from the 6th up to the 15th day; at this time, they were evident in about 70% of fibroblastic cells (myofibroblasts). They then regressed progressively, and on the 30th day, microfilaments were no longer present in scar fibroblasts. alpha-Smooth muscle actin, but not smooth muscle myosin and desmin, was always present in microfilament bundles of myofibroblasts. The staining intensity was progressive from the 6th to the 15th day and decreased thereafter; no staining was seen at the 30th day. Between the 20th and the 25th day, many apoptotic figures were seen in fibroblastic and endothelial cells, suggesting that apoptosis is the mechanism of their disappearance. We conclude that myofibroblasts develop gradually from granulation tissue fibroblasts and temporarily express a marker of smooth muscle differentiation. These results may be relevant for the understanding of the mechanisms of normal and pathologic wound healing.