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Bullous pemphigoid (BP) is an autoimmune bullous disease, associated with autoantibodies directed against the hemidesmosomal components BP180 and BP230. In this study for the first time different laboratories have analyzed the autoantibody profile in the same group of 49 prospectively recruited BP patients. The results show that: 1) disease severity and activity correlated with levels of IgG against the BP180-NC16A domain, but also against a COOH-terminal epitope of BP180, 2) distinct epitopes of the BP180 ectodomain other than BP180-NC16A were recognized by 96% of the BP sera; and 3) the combined use of BP180 and BP230 ELISA led to the detection of IgG autoantibodies in all the BP sera. These results demonstrate the usefulness of the combined ELISAs based on various BP180 and BP230 fragments in establishing the diagnosis of BP and support the concept that BP180 is the major autoantigen of BP.

ApoA-1 (apolipoprotein A-1) is the main component of HDL (high-density lipoprotein) and stabilizes PON-1 (paraoxonase-1), which prevents lipid peroxidation and oxLDL (oxidized low-density lipoprotein) formation. Autoantibodies against apoA-1 [anti-(apoA-1) IgG] have been found in antiphospholipid syndrome and systemic lupus erythematosous, two diseases with an increased risk of thrombotic events, as well as in ACS (acute coronary syndrome). OxLDL levels are also elevated in these diseases. Whether anti-(apoA-1) IgGs exist in other prothrombotic conditions, such as APE (acute pulmonary embolism) and stroke, has not been studied and their potential association with oxLDL and PON-1 activity is not known. In the present study, we determined prospectively the prevalence of anti-(apoA-1) IgG in patients with ACS (n=127), APE (n=58) and stroke (n=34), and, when present, we tested their association with oxLDL levels. The prevalance of anti-(apoA-1) IgG was 11% in the ACS group, 2% in the control group and 0% in the APE and stroke groups. The ACS group had significantly higher median anti-(apoA-1) IgG titres than the other groups of patients. Patients with ACS positive for anti-(apoA-1) IgG had significantly higher median oxLDL values than those who tested negative (226.5 compared with 47.7 units/l; P

Myotilin and the calsarcin family member FATZ-1 (also called calsarcin-2 or myozenin-1) are recently discovered sarcomeric proteins implicated in the assembly and stabilization of the Z-discs in skeletal muscle. The essential role of myotilin in skeletal muscle is attested by the observation that certain forms of myofibrillar myopathy and limb girdle muscular dystrophy are caused by mutations in the human myotilin gene. Here we show by transfection, biochemical and/or yeast two-hybrid assay that: (1) myotilin is able to interact with the C-terminal region of FATZ-1 and that the N- or C-terminal truncations of myotilin abrogate binding; (2) myotilin can also interact with another calsarcin member, FATZ-2 (calsarcin-1, myozenin-2); (3) myotilin and FATZ-1 bind not only to the C-terminal region of filamin-C containing the Ig repeats 19-24, but also to the other two filamins, filamin-A and filamin-B, as well as the newly identified filamin-Bvar-1variant; (4) the binding of myotilin to filamin-C involves binding sites in its N-terminal region, whereas FATZ-1 associates with filamin-C via sequences within either its N- or C-terminal region; and finally, (5) the C-terminal region of filamin-C like filamin-B and filamin-Bvar-1, shows binding activity with the β1A integrin subunit. Our findings further dissect the molecular interactions within the Z-disc that are essential for its organization, and provide evidence for a novel connection between Z-disc proteins and the sarcolemma via filamins and β1 integrins. These data shed new light on the complex organization of the Z-disc that is highly relevant to understanding muscular dystrophies.

Summary Background ERBIN is a binding partner of Erb-B2, an orphan receptor within the Erb-B family critically involved in the regulation of cell growth and differentiation. Although its function remains unclear, ERBIN is thought to affect the polarity of epithelial cells and cell growth via the Ras signalling pathway. Objectives To examine and compare the tissue distribution and the expression levels of ERBIN and Erb-B2 in normal skin and in cutaneous carcinomas. Methods Fifteen cases of basal cell carcinoma (BCC), 12 cases of squamous cell carcinoma (SCC) and five cases of keratoacanthoma (KA) were analysed by immunohistochemistry on paraffin-embedded sections using anti-ERBIN and anti-Erb-B2 antibodies. Results ERBIN and Erb-B2 had a similar distribution in normal human skin. They were primarily localized at the plasma membrane in differentiated keratinocytes and in duct cells from eccrine glands, whereas they were localized diffusely in the cytoplasma of basal keratinocytes. In both SCC and KA the subcellular distribution of ERBIN and Erb-B2 remained unchanged, whereas both proteins were redistributed from the plasma membrane into cytosolic aggregates in BCC. Conclusions The subcellular localization of ERBIN in normal human skin is similar to that of Erb-B2 and varies with cell differentiation. Based on our findings and on the biological activities of Erb-B2, it is conceivable that disturbed expression or functioning of ERBIN and Erb-B2 is implicated in the development of the malignant phenotype of BCC.

Publisher Summary This chapter describes two very different approaches for investigating intermediate filament– Desmoplakins (IF–DP) interactions—in vitro purification/cosedimentation and yeast three-hybrid (Y3H) analyses—and discusses functional assays for determining the mechanical properties of epithelial cell sheets harboring mutant DPs that compromise the IF–desmosome connection. The various approaches that have been used to address the question of IF–DP interactions have provided data that are frequently in agreement, but differ in certain areas, for example, the importance of the flexible C-terminal tail in DP has been confirmed in multiple studies. . The chapter presents an assay that was adapted for studying the effects of engineered mutations and cells derived from patients with naturally occurring truncations of DP. This assay can also be used to study the effects of pharmacological agents, blocking antibodies, and mutations in other plaque and transmembrane proteins on adhesive strength. The methods described in this chapter provide a toolbox to understand both the molecular requirements for establishing an IF–desmosome connection, and for analyzing the functional effects of perturbing this interaction on the integrity of epithelial cell sheets.

The bullous pemphigoid antigen 1 (BP230) and desmoplakin (DP) are members of the plakin protein family of cytolinkers. Despite their homology, their COOH termini selectively bind distinct intermediate filaments (IFs). We studied sequences within their COOH termini required for their interaction with the epidermal keratins K5/K14, the simple epithelial keratins K8/K18, and type III IF vimentin by yeast three-hybrid, cell transfection, and overlay assays. The results indicate that BP230 interacts with K5/K14 but not with K8/K18 or vimentin via a region encompassing both the B and C subdomains and the COOH extremity, including a COOH-terminal eight-amino-acid stretch. In contrast, the C subdomain with the COOH-terminal extremity of DP interacts with K5/K14 and K8/K18, and its linker region is able to associate with K8/K18 and vimentin. Furthermore, the potential of DP to interact with IF proteins in yeast seems to be regulated by phosphorylation of Ser 2849 within its COOH terminus. Strikingly, BP230 and DP interacted with cytokeratins only when both type I and type II keratins were present. The head and tail domains of K5/K14 keratins were dispensable for their interaction with BP230 or DP. On the basis of our findings, we postulate that (1) the binding specificity of plakins for various IF proteins depends on their linker region between the highly homologous B and C subdomains and their COOH extremity and (2) the association of DP and BP230 with both epidermal and simple keratins is critically affected by the tertiary structure induced by heterodimerization and involves recognition sites located primarily in the rod domain of these keratins.

This article review summarizes data on cell-substratum adhesion complexes involved in the regulation of cellular functions in the intestine. We first focus on the molecular composition of the two main adhesion structures-the beta1 integrin-adhesion complex and the hemidesmosome-found in vivo and in two human intestinal cell lines. We also report the key findings on the cellular behavior and response to the extracellular matrix that involve integrins, the main transmembrane anchors of these complexes. How the dynamics of cell/extracellular matrix interactions contribute to cell migration, proliferation, differentiation, and tumorigenicity is discussed in the light of the data provided by the human intestinal cells.

Recently, we have shown that a region within the beta4 cytoplasmic domain, encompassing the second fibronectin type III (FNIII) repeat and the first 27 amino acids of the connecting segment, is critical for the localization of alpha6 beta4 in hemidesmosomes. In addition, this region was shown to regulate the distribution of HD1/plectin in transfected cells. In order to investigate the function of the beta4 extracellular and cytoplasmic domains in the assembly and integrity of hemidesmosomes, we have constructed chimeric receptors consisting of the extracellular and transmembrane domains of the interleukin 2 receptor (IL2R), fused to different parts of the beta4 cytoplasmic domain. These chimeras are expressed as single subunits at the plasma membrane. The results show that the first and the second FNIII repeat, together with the first part of the connecting segment (in total a stretch of 241 amino acids spanning amino acids 1,115 to 1,356) are both essential and sufficient for the localization of beta4 in pre-existing hemidesmosomes. Moreover, expression of the IL2R/beta4 chimeric constructs in COS-7 and CHO cells, which do not express alpha6 beta4 or the bullous pemphigoid (BP) antigens but do express HD1/plectin, revealed that the stretch of 241 amino acids is sufficient for inducing the formation of type II hemidesmosomes. Expression of the IL2R/beta4 chimeras in a keratinocyte cell line derived from a patient lacking beta4 expression, showed that amino acids 1,115 to 1,356 can also induce the formation of type I hemidesmosomes. We further demonstrate that type I and II hemidesmosomes can also be formed upon adhesion of alpha6 beta4-expressing cells to fibronectin. These findings establish that the beta4 extracellular domain is not essential for the induction of hemidesmosome assembly. Moreover, they demonstrate that binding of alpha6 beta4 to ligand, and heterodimerization of alpha6 with beta4, are not required for hemidesmosome formation. This indicates that the assembly of hemidesmosomes can be regulated from within the cell.

Bullous pemphigoid (BP), the most common autoimmune subepidermal bullous disease, is associated with an autoantibody response to BP180 and BP230, two components of junctional adhesion complexes in human skin promoting dermo-epidermal cohesion. Retrospective analyses demonstrated that these autoantigens harbor several epitopes targeted by autoaggressive B and T cells. The aim of this prospective multicenter study was to assess the evolution of IgG autoantibodies in 35 BP patients over a 12-month observation period. Epitope-spreading (ES) events were detected in 17 of 35 BP patients (49%). They preferentially occurred in an early stage of the disease and were significantly related to disease severity at diagnosis. Moreover, in three patients, spreading of IgG reactivity to intracellular epitopes of BP180 and BP230 was preceded by recognition of the BP180 ectodomain. Finally, IgG reactivity with extracellular epitopes of BP180 and intracellular epitopes of BP230 correlated with the severity of BP in disease course. These findings support the idea that IgG recognition of the BP180 ectodomain is an early and crucial event in BP disease, followed by variable intra- and intermolecular ES events, which likely shape the individual course of BP.

Plectin, a cytolinker of the plakin family, anchors the intermediate filament (IF) network formed by keratins 5 and 14 (K5/K14) to hemidesmosomes, junctional adhesion complexes in basal keratinocytes. Genetic alterations of these proteins cause epidermolysis bullosa simplex (EBS) characterized by disturbed cytoarchitecture and cell fragility. The mechanisms through which mutations located after the documented plectin IF-binding site, composed of the plakin-repeat domain (PRD) B5 and the linker, as well as mutations in K5 or K14, lead to EBS remain unclear. We investigated the interaction of plectin C terminus, encompassing four domains, the PRD B5, the linker, the PRD C, and the C extremity, with K5/K14 using different approaches, including a rapid and sensitive fluorescent protein–binding assay, based on enhanced green fluorescent protein-tagged proteins (FluoBACE). Our results demonstrate that all four plectin C-terminal domains contribute to its association with K5/K14 and act synergistically to ensure efficient IF binding. The plectin C terminus predominantly interacted with the K5/K14 coil 1 domain and bound more extensively to K5/K14 filaments compared with monomeric keratins or IF assembly intermediates. These findings indicate a multimodular association of plectin with K5/K14 filaments and give insights into the molecular basis of EBS associated with pathogenic mutations in plectin, K5, or K14 genes.

Plectin is a major component of the cytoskeleton and is expressed in a wide variety of cell types. It plays an important role in the integrity of the cytoskeleton by cross-linking the three filamentous networks and stabilizing cell-matrix and cell-cell contacts. Sequence analysis showed that plectin contains a highly conserved actin-binding domain, consisting of a pair of calponin-like subdomains. Using yeast two-hybrid assays in combination with in vitro binding experiments, we demonstrate that the actin-binding domain of plectin is fully functional and preferentially binds to polymeric actin. The sequences required for actin binding were identified at the C-terminal end of the first calponin homology domain within the actin-binding domain of plectin. We found that the actin-binding domain of plectin is able to bundle actin filaments and we present evidence that this is mediated by the dimerization of this domain. In addition we also show that plectin and another member of the plakin family, dystonin, can heterodimerize by their actin-binding domains. We propose a new mechanism by which plectin and possibly also other actin-binding proteins can regulate the organization of the F-actin network in the cell.

Transforming growth factor beta (TGFbeta), the most established promoter of myofibroblast differentiation, induces ED-A cellular fibronectin and alpha-smooth muscle actin expression in fibroblastic cells in vivo and in vitro. ED-A fibronectin exerts a permissive action for alpha-smooth muscle actin expression. A morphological continuity (called fibronexus), a specialized form of focal adhesion, has been described between actin stress fibers that contain alpha-smooth muscle actin, and extracellular fibronectin, which contains the ED-A portion, in both cultured fibroblasts and granulation tissue myofibroblasts. We have studied the development of these focal adhesions in TGFbeta-treated fibroblasts using confocal laser scanning microscopy, three-dimensional image reconstruction and western blots using antibodies against focal adhesion proteins. The increase in ED-A fibronectin expression induced by TGFbeta was accompanied by bundling of ED-A fibronectin fibers and their association with the terminal portion of alpha-smooth muscle actin-positive stress fibers. In parallel, the focal adhesion size was importantly increased, and tensin and FAK were neoexpressed in focal adhesions; moreover, vinculin and paxillin were recruited from the cytoplasmic pool into focal adhesions. We have evaluated morphometrically the length and area of focal adhesions. In addition, we have evaluated biochemically their content of associated proteins and of alpha-smooth muscle actin after TGFbeta stimulation and on this basis suggest a new focal adhesion classification, that is, immature, mature and supermature. When TGFbeta-induced alpha-smooth muscle actin expression was blocked by soluble recombinant ED-A fibronectin, we observed that the fragment was localised into the fibronectin network at the level of focal adhesions and that focal adhesion supermaturation was inhibited. The same effect was also exerted by the ED-A fibronectin antibody IST-9. In addition, the antagonists of actin-myosin contractility BDM and ML-7 provoked the dispersion of focal adhesions and the decrease of alpha-smooth muscle actin content in stress fibers of pulmonary fibroblasts, which constitutively show large focal adhesions and numerous stress fibers that contain alpha-smooth muscle actin. These inhibitors also decreased the incorporation of recombinant ED-A into fibronectin network. Our data indicate that a three-dimensional transcellular structure containing both ED-A fibronectin and alpha-smooth muscle actin plays an important role in the establishment and modulation of the myofibroblastic phenotype. The organisation of this structure is regulated by intracellularly and extracellularly originated forces.