Tetraspanin CD151 and integrin α3ß1 contribute to the stabilization of integrin α6ß4-containing cell-matrix adhesions.

Tetraspanin CD151 has been suggested to regulate cell adhesion through its association with laminin-binding integrins α3ß1 and α6ß4; however, its precise function in keratinocyte adhesion remains elusive. In this study, we investigated the role of CD151 in the formation and maintenance of laminin-associated adhesions. We show that CD151, through binding to integrin α3ß1, plays a critical role in the stabilization of an adhesion structure with a distinct molecular composition of hemidesmosomes with tetraspanin features. These hybrid cell-matrix adhesions, which are formed early during cell adhesion and spreading and at later stages of cell spreading, are present in the central region of the cells. They contain the CD151-α3ß1/α6ß4 integrin complexes and the cytoskeletal linker protein plectin, but are not anchored to the keratin filaments. In contrast, hemidesmosomes, keratin filament-associated adhesions that contain integrin α6ß4, plectin, BP180 (encoded by COL17A1) and BP230 (encoded by DST), do not require CD151 for their formation or maintenance. These findings provide new insights into the dynamic and complex regulation of adhesion structures in keratinocytes and the pathogenic mechanisms underlying skin blistering diseases caused by mutations in the gene for CD151.

Integrin α6ß4 Recognition of a Linear Motif of Bullous Pemphigoid Antigen BP230 Controls Its Recruitment to Hemidesmosomes.

Mechanical stability of epithelia requires firm attachment to the basement membrane via hemidesmosomes. Dysfunction of hemidesmosomal proteins causes severe skin-blistering diseases. Two plakins, plectin and BP230 (BPAG1e), link the integrin α6ß4 to intermediate filaments in epidermal hemidesmosomes. Here, we show that a linear sequence within the isoform-specific N-terminal region of BP230 binds to the third and fourth FnIII domains of ß4. The crystal structure of the complex and mutagenesis analysis revealed that BP230 binds between the two domains of ß4. BP230 induces closing of the two FnIII domains that are locked in place by an interdomain ionic clasp required for binding. Disruption of BP230-ß4 binding prevents recruitment of BP230 to hemidesmosomes in human keratinocytes, revealing a key role of this interaction for hemidesmosome assembly. Phosphomimetic substitutions in ß4 and BP230 destabilize the complex. Thus, our study provides insights into the architecture of hemidesmosomes and potential mechanisms of regulation.

A systematic survey of conformational states in ß1 and ß4 integrins using negative-stain electron microscopy.

Structural analyses of ß2 and ß3 integrins have revealed that they generally assume a compact bent conformation in the resting state and undergo a global conformational transition involving extension during upregulation of ligand affinity, collectively called the 'switchblade model'. This hypothesis, however, has not been extensively tested for other classes of integrins. We prepared a set of recombinant integrin ectodomain fragments including αvß3, α2ß1, α3ß1, α5ß1, α6ß1 and α6ß4, and used negative-stain electron microscopy to examine their structures under various conditions. In contrast to αvß3 integrin, which exhibited a severely bent conformation in low-affinity 5 mM Ca2+ conditions, all ß1 integrin heterodimers displayed a mixed population of half-bent to fully extended conformations. Moreover, they did not undergo significant conformational change upon activation by Mn2+ Integrin α6ß4 was even more resistant to conformational regulation, showing a completely extended structure regardless of the buffer conditions. These results suggest that the mechanisms of conformational regulation of integrins are more diverse and complex than previously thought, requiring more experimental scrutiny for each integrin subfamily member.

Radioimmunotherapy of pancreatic cancer xenografts in nude mice using 90Y-labeled anti-α6ß4 integrin antibody.

The contribution of integrin α6ß4 (α6ß4) overexpression to the pancreatic cancer invasion and metastasis has been previously shown. We have reported immunotargeting of α6ß4 for radionuclide-based and near-infrared fluorescence imaging in a pancreatic cancer model. In this study, we prepared yttrium-90 labeled anti-α6ß4 antibody (90Y-ITGA6B4) and evaluated its radioimmunotherapeutic efficacy against pancreatic cancer xenografts in nude mice. Mice bearing xenograft tumors were randomly divided into 5 groups: (1) single administration of 90Y-ITGA6B4 (3.7MBq), (2) double administrations of 90Y-ITGA6B4 with once-weekly schedule (3.7MBq x 2), (3) single administration of unlabeled ITGA6B4, (4) double administrations of unlabeled ITGA6B4 with once-weekly schedule and (5) the untreated control. Biweekly tumor volume measurements and immunohistochemical analyses of tumors at 2 days post-administration were performed to monitor the response to treatments. To assess the toxicity, body weight was measured biweekly. Additionally, at 27 days post-administration, blood samples were collected through cardiac puncture, and hematological parameters, hepatic and renal functions were analyzed. Both 90Y-ITGA6B4 treatment groups showed reduction in tumor volumes (P < 0.04), decreased cell proliferation marker Ki-67-positive cells and increased DNA damage marker p-H2AX-positive cells, compared with the other groups. Mice treated with double administrations of 90Y-ITGA6B4, exhibited myelosuppression. There were no significant differences in hepatic and renal functions between the 2 treatment groups and the other groups. Our results suggest that 90Y-ITGA6B4 is a promising radioimmunotherapeutic agent against α6ß4 overexpressing tumors. In the future studies, dose adjustment for fractionated RIT should be considered carefully in order to get the optimal effect while avoiding myelotoxicity.

Improved Synthesis and In Vitro Evaluation of an Aptamer Ribosomal Toxin Conjugate.

Delivery of toxins, such as the ricin A chain, Pseudomonas exotoxin, and gelonin, using antibodies has had some success in inducing specific toxicity in cancer treatments. However, these antibody-toxin conjugates, called immunotoxins, can be bulky, difficult to express, and may induce an immune response upon in vivo administration. We previously reported delivery of a recombinant variant of gelonin (rGel) by the full-length prostate-specific membrane antigen (PSMA) binding aptamer, A9, to potentially circumvent some of these problems. Here, we report a streamlined approach to generating aptamer-rGel conjugates utilizing a chemically synthesized minimized form of the A9 aptamer. Unlike the full-length A9 aptamer, this minimized variant can be chemically synthesized with a 5' terminal thiol. This facilitates the large scale synthesis and generation of aptamer toxin conjugates linked by a reducible disulfide linkage. Using this approach, we generated aptamer-toxin conjugates and evaluated their binding specificity and toxicity. On PSMA(+) LNCaP prostate cancer cells, the A9.min-rGel conjugate demonstrated an IC50 of ∼60 nM. Additionally, we performed a stability analysis of this conjugate in mouse serum where the conjugate displayed a t1/2 of ∼4 h, paving the way for future in vivo experiments.

Cytoplasmic domain interactions of syndecan-1 and syndecan-4 with α6ß4 integrin mediate human epidermal growth factor receptor (HER1 and HER2)-dependent motility and survival.

Epithelial cells are highly dependent during wound healing and tumorigenesis on the α6ß4 integrin and its association with receptor tyrosine kinases. Previous work showed that phosphorylation of the ß4 subunit upon matrix engagement depends on the matrix receptor syndecan (Sdc)-1 engaging the cytoplasmic domain of the ß4 integrin and coupling of the integrin to human epidermal growth factor receptor-2 (HER2). In this study, HER2-dependent migration activated by matrix engagement is compared with migration stimulated by EGF. We find that whereas HER2-dependent migration depends on Sdc1, EGF-dependent migration depends on a complex consisting of human epidermal growth factor receptor-1 (HER1, commonly known as EGFR), α6ß4, and Sdc4. The two syndecans recognize distinct sites at the extreme C terminus of the ß4 integrin cytoplasmic domain. The binding motif in Sdc1 is QEEXYX, composed in part by its syndecan-specific variable (V) region and in part by the second conserved (C2) region that it shares with other syndecans. A cell-penetrating peptide containing this sequence competes for HER2-dependent epithelial migration and carcinoma survival, although it is without effect on the EGFR-stimulated mechanism. ß4 mutants bearing mutations specific for Sdc1 and Sdc4 recognition act as dominant negative mutants to block cell spreading or cell migration that depends on HER2 or EGFR, respectively. The interaction of the α6ß4 integrin with the syndecans appears critical for it to be utilized as a signaling platform; migration depends on α3ß1 integrin binding to laminin 332 (LN332; also known as laminin 5), whereas antibodies that block α6ß4 binding are without effect. These findings indicate that specific syndecan family members are likely to have key roles in α6ß4 integrin activation by receptor tyrosine kinases.

Cross-talk between integrin α6ß4 and insulin-like growth factor-1 receptor (IGF1R) through direct α6ß4 binding to IGF1 and subsequent α6ß4-IGF1-IGF1R ternary complex formation in anchorage-independent conditions.

Integrin αvß3 plays a role in insulin-like growth factor-1 (IGF1) signaling (integrin-IGF1 receptor (IGF1R) cross-talk). The specifics of the cross-talk are, however, unclear. In a current model, "ligand occupancy" of αvß3 (i.e. the binding of extracellular matrix proteins) enhances signaling induced by IGF1 binding to IGF1R. We recently reported that IGF1 directly binds to αvß3 and induces αvß3-IGF1-IGF1R ternary complex formation. Consistently, the integrin binding-defective IGF1 mutant (R36E/R37E) is defective in inducing ternary complex formation and IGF signaling, but it still binds to IGF1R. Like αvß3, integrin α6ß4 is overexpressed in many cancers and is implicated in cancer progression. Here, we discovered that α6ß4 directly bound to IGF1, but not to R36E/R37E. Grafting the ß4 sequence WPNSDP (residues 167-172), which corresponds to the specificity loop of ß3, to integrin ß1 markedly enhanced IGF1 binding to ß1, suggesting that the WPNSDP sequence is involved in IGF1 recognition. WT IGF1 induced α6ß4-IGF1-IGF1R ternary complex formation, whereas R36E/R37E did not. When cells were attached to matrix, exogenous IGF1 or α6ß4 expression had little or no effect on intracellular signaling. When cell-matrix adhesion was reduced (in poly(2-hydroxyethyl methacrylate-coated plates), IGF1 induced intracellular signaling and enhanced cell survival in an α6ß4-dependent manner. Also IGF1 enhanced colony formation in soft agar in an α6ß4-dependent manner. These results suggest that IGF binding to α6ß4 plays a major role in IGF signaling in anchorage-independent conditions, which mimic the in vivo environment, and is a novel therapeutic target.

Integrin (alpha6beta4) signals through Src to increase expression of S100A4, a metastasis-promoting factor: implications for cancer cell invasion.

Integrin alpha6beta4 is linked to cancer cell motility and invasion in aggressive and metastatic cancer cells. In this study, we showed that expression of the beta4 integrin in MDA-MB-435 cancer cells (MDA-MB-435/beta4) leads to a dramatic increase in expression of a metastasis-promoting factor, S100A4, as determined by affymetrix gene chip microarray, quantitative real-time PCR, and Western blot analysis. Alternatively, knocking down beta4 integrin expression in MDA-MB-231 breast carcinoma cells by shRNA reduced the level of S100A4 expression. The mechanism by which alpha6beta4 enhances S100A4 expression involves Src, Akt, and NFAT. We have further shown that Y1494, a tyrosine residue of the ITIM motif in the cytoplasmic domain of the beta4 integrin subunit, is essential for alpha6beta4-dependent S100A4 expression. Reduction of S100A4 expression by shRNA blocked migration, invasion, and anchorage-independent growth of MDA-MB-435/beta4, SUM-159, and MDA-MB-231 cells. These studies define a novel mechanism by which integrin alpha6beta4 promotes cancer cell motility and invasion, and provides insight into how S100A4 expression is regulated in cancer cells.

Structural basis of the interaction between integrin alpha6beta4 and plectin at the hemidesmosomes.

The interaction between the integrin alpha6beta4 and plectin is essential for the assembly and stability of hemidesmosomes, which are junctional adhesion complexes that anchor epithelial cells to the basement membrane. We describe the crystal structure at 2.75 A resolution of the primary alpha6beta4-plectin complex, formed by the first pair of fibronectin type III domains and the N-terminal region of the connecting segment of beta4 and the actin-binding domain of plectin. Two missense mutations in beta4 (R1225H and R1281W) linked to nonlethal forms of epidermolysis bullosa prevent essential intermolecular contacts. We also present two structures at 1.75 and 2.05 A resolution of the beta4 moiety in the absence of plectin, which reveal a major rearrangement of the connecting segment of beta4 on binding to plectin. This conformational switch is correlated with the way alpha6beta4 promotes stable adhesion or cell migration and suggests an allosteric control of the integrin.

A key tyrosine (Y1494) in the beta4 integrin regulates multiple signaling pathways important for tumor development and progression.

Expression of the alpha6beta4 integrin is associated with poor patient prognosis and reduced survival in a variety of human cancers. In recent years, a limited number of in vivo studies have examined the contribution of this integrin receptor to cancer progression and they have revealed that the alpha6beta4 integrin plays a multifaceted role in regulating tumor development and progression. In the current study, we investigated the mechanism by which one tyrosine residue in the beta4 subunit cytoplasmic domain, Y1494, contributes to the tumor-promoting functions of the alpha6beta4 integrin in vivo. We show that Y1494 participates in the stimulation of diverse signaling pathways that promote alpha6beta4-dependent tumor growth and invasion. Mutation of Y1494 inhibits the ability of the alpha6beta4 integrin to support anchorage-independent growth in vitro and tumor development and angiogenesis in vivo, a result that mimics the loss of total expression of the beta4 subunit. Our results support the hypothesis that Y1494 regulates alpha6beta4-dependent anchorage-independent growth through activation of the extracellular signal-regulated kinase 1/2 signaling pathway, and invasion through the combined activation of phosphatidylinositol 3-kinase and Src. Collectively, our results identify Y1494 as a major regulatory site for signaling from the alpha6beta4 integrin to promote tumor development and progression.

Regulation of hemidesmosome disassembly by growth factor receptors.

Hemidesmosomes (HDs) promote the stable adhesion of basal epithelial cells to the underlying basement membrane (BM). Critical for the mechanical stability of the HD is the interaction between integrin alpha6beta4 and plectin, which is destabilized when HD disassembly is required, for instance, to allow keratinocyte migration during wound healing. Growth factors such as epidermal growth factor (EGF) can trigger HD disassembly and induce phosphorylation of the beta4 intracellular domain. Whereas tyrosine phosphorylation appears to mediate cooperation with growth factor signaling pathways and invasion in carcinoma cells, serine phosphorylation seems the predominant mechanism for regulating HD destabilization. Here, we discuss recent advances that shed light on the residues involved, the identity of the kinases that phosphorylate them, and the interactions that become disrupted by these phosphorylations.

Peripheral myelin protein 22 is in complex with alpha6beta4 integrin, and its absence alters the Schwann cell basal lamina.

Peripheral myelin protein 22 (PMP22) is a tetraspan membrane glycoprotein, the misexpression of which is associated with hereditary demyelinating neuropathies. Myelinating Schwann cells (SCs) produce the highest levels of PMP22, yet the function of the protein in peripheral nerve biology is unresolved. To investigate the potential roles of PMP22, we engineered a novel knock-out (-/-) mouse line by replacing the first two coding exons of pmp22 with the lacZ reporter. PMP22-deficient mice show strong beta-galactosidase reactivity in peripheral nerves, cartilage, intestines, and lungs, whereas phenotypically they display the characteristics of tomaculous neuropathy. In the absence of PMP22, myelination of peripheral nerves is delayed, and numerous axon-SC profiles show loose basal lamina, suggesting altered interactions of the glial cells with the extracellular matrix. The levels of beta4 integrin, a molecule involved in the linkage between SCs and the basal lamina, are severely reduced in nerves of PMP22-deficient mice. During early stages of myelination, PMP22 and beta4 integrin are coexpressed at the cell surface and can be coimmunoprecipitated together with laminin and alpha6 integrin. In agreement, in clone A colonic carcinoma cells, epitope-tagged PMP22 forms a complex with beta4 integrin. Together, these data indicate that PMP22 is a binding partner in the integrin/laminin complex and is involved in mediating the interaction of SCs with the extracellular environment.

Tyrosine kinase signal specificity: lessons from the HGF receptor.

There are two major lines of thinking concerning the mechanisms responsible for specificity in receptor tyrosine kinase signalling. On one hand, receptors might provide instructive signals that dictate cell-fate decisions and, on the other, they might generate permissive signals unleashing responses that are inherently defined in the protein repertoire of target cells. Recent data indicate that the signalling activity of the Met receptor for hepatocyte growth factor is affected by association with cell-specific surface molecules, namely the alpha6beta4 integrin, Plexin B1 and CD44. This suggests that integration of cell-restricted expression of receptor partners that modulate kinase outputs with the intrinsic signalling features of receptors is required for specification of biological responses.

Dynamics of the alpha6beta4 integrin in keratinocytes.

The integrin alpha6beta4 has been implicated in two apparently contrasting processes, i.e., the formation of stable adhesions, and cell migration and invasion. To study the dynamic properties of alpha6beta4 in live cells two different beta4-chimeras were stably expressed in beta4-deficient PA-JEB keratinocytes. One chimera consisted of full-length beta4 fused to EGFP at its carboxy terminus (beta4-EGFP). In a second chimera the extracellular part of beta4 was replaced by EGFP (EGFP-beta4), thereby rendering it incapable of associating with alpha6 and thus of binding to laminin-5. Both chimeras induce the formation of hemidesmosome-like structures, which contain plectin and often also BP180 and BP230. During cell migration and division, the beta4-EGFP and EGFP-beta4 hemidesmosomes disappear, and a proportion of the beta4-EGFP, but not of the EGFP-beta4 molecules, become part of retraction fibers, which are occasionally ripped from the cell membrane, thereby leaving "footprints" of the migrating cell. PA-JEB cells expressing beta4-EGFP migrate considerably more slowly than those that express EGFP-beta4. Studies with a beta4-EGFP mutant that is unable to interact with plectin and thus with the cytoskeleton (beta4(R1281W)-EGFP) suggest that the stabilization of the interaction between alpha6beta4 and LN-5, rather than the increased adhesion to LN-5, is responsible for the inhibition of migration. Consistent with this, photobleaching and recovery experiments revealed that the interaction of beta4 with plectin renders the bond between alpha6beta4 and laminin-5 more stable, i.e., beta4-EGFP is less dynamic than beta4(R1281W)-EGFP. On the other hand, when alpha6beta4 is bound to laminin-5, the binding dynamics of beta4 to plectin are increased, i.e., beta4-EGFP is more dynamic than EGFP-beta4. We suggest that the stability of the interaction between alpha6beta4 and laminin-5 is influenced by the clustering of alpha6beta4 through the deposition of laminin-5 underneath the cells. This clustering ultimately determines whether alpha6beta4 will inhibit cell migration or not.