Kindlin-1 regulates keratinocyte electrotaxis

Kindler syndrome (KS) is an autosomal recessive blistering skin disease resulting from pathogenic mutations in FERMT1. This gene encodes kindlin-1, a focal adhesion protein involved in activation of the integrin family of extracellular matrix receptors. Most cases of KS show a marked reduction or complete absence of the kindlin-1 protein in keratinocytes, resulting in defective cell adhesion and migration. Electric fields also act as intrinsic regulators of adhesion and migration in the skin, but the molecular mechanisms by which this occurs are poorly understood. Here we show that keratinocytes derived from KS patients are unable to undergo electrotaxis, and this defect is restored by overexpression of wild-type kindlin-1 but not a W612A mutation that prevents kindlin-integrin binding. Moreover, deletion of the pleckstrin homology domain of kindlin-1 also failed to rescue electrotaxis in KS cells, indicating that both integrin and lipid binding are required for this function. Kindlin-1 was also required for the maintenance of lamellipodial protrusions during electrotaxis via electric field-activated β1 integrin. Indeed, inhibition of β1 integrins also leads to loss of electrotaxis in keratinocytes. Our data suggest that loss of kindlin-1 function may therefore result in epithelial insensitivity to electric fields and contribute to KS disease pathology

Drug-repositioning screens identify Triamterene as a selective drug for the treatment of DNA Mismatch Repair deficient cells.

Purpose: The DNA Mismatch repair (MMR) pathway is required for the maintenance of genome stability. Unsurprisingly, mutations in MMR genes occur in a wide range of different cancers. Studies thus far have largely focused on specific tumor types or MMR mutations, however it is becoming increasingly clear that a therapy targeting MMR-deficiency in general would be clinically very beneficial. Experimental Design: Based on a drug-repositioning approach, we screened a large panel of cell lines with various MMR deficiencies from a range of different tumor types with a compound drug library of previously approved drugs. We have identified the potassium-sparing diuretic drug Triamterene, as a novel sensitizing agent in MMR-deficient tumor cells, in vitro and in vivo. Results: The selective tumor cell cytotoxicity of Triamterene occurs through its antifolate activity, and depends on the activity of the folate synthesis enzyme, thymidylate synthase. Triamterene leads to a thymidylate synthase-dependent differential increase in reactive oxygen species in MMR-deficient cells, ultimately resulting in an increase in DNA double strand breaks. Conclusion: Conclusively, our data reveal a new drug repurposing and novel therapeutic strategy that has potential for the treatment of MMR-deficiency in a range of different tumor types and could significantly improve patient survival

Germline Mutation in EXPH5 Implicates the Rab27B Effector Protein Slac2-b in Inherited Skin Fragility

The Rab GTPase Rab27B and one of its effector proteins, Slac2-b (also known as EXPH5, exophilin-5), have putative roles in intracellular vesicle trafficking but their relevance to human disease is not known. By using whole-exome sequencing, we identified a homozygous frameshift mutation in EXPH5 in three siblings with inherited skin fragility born to consanguineous Iraqi parents. All three individuals harbor the mutation c.5786delC (p.Pro1929Leufs∗8) in EXPH5, which truncates the 1,989 amino acid Slac2-b protein by 52 residues. The clinical features comprised generalized scale-crusts and occasional blisters, mostly induced by trauma, as well as mild diffuse pigmentary mottling on the trunk and proximal limbs. There was no increased bleeding tendency, no neurologic abnormalities, and no increased incidence of infection. Analysis of an affected person's skin showed loss of Slac2-b immunostaining (C-terminal antibody), disruption of keratinocyte adhesion within the lower epidermis, and an increased number of perinuclear vesicles. A role for Slac2-b in keratinocyte biology was supported by findings of cytoskeletal disruption (mainly keratin intermediate filaments) and decreased keratinocyte adhesion in both keratinocytes from an affected subject and after shRNA knockdown of Slac2-b in normal keratinocytes. Slac2-b was also shown to colocalize with Rab27B and β4 integrin to early adhesion initiation sites in spreading normal keratinocytes. Collectively, our findings identify an unexpected role for Slac2-b in inherited skin fragility and expand the clinical spectrum of human disorders of GTPase effector proteins

BPAG1-e restricts keratinocyte migration through control of adhesion stability

Bullous pemphigoid antigen 1 (BPAG1-e, also known as BP230) is a member of the plakin family of hemidesmosome cytoskeletal linker proteins that is encoded by an isoform of the dystonin (DST) gene. Recently, we reported two unrelated families with homozygous nonsense mutations in this DST isoform that led to ultrastructural loss of hemidesmosomal inner plaques and clinical features of trauma-induced skin fragility. We now demonstrate that keratinocytes isolated from these individuals have significant defects in adhesion, as well as increased cell spreading and migration. These mutant keratinocytes also display reduced levels of β4 integrins at the cell surface but increased total protein levels of keratin-14 and β1 integrins. These alterations in cell behavior and protein expression were not seen in control keratinocytes in which BPAG1-e expression had been silenced by stable expression of short hairpin RNA to target DST. The failure of knockdown approaches to recapitulate the changes in morphology, adhesion, and migration seen in patient cells therefore suggests such approaches are not appropriate to study loss of this protein in vivo. The contrasting findings in keratinocytes harboring naturally occurring mutations, however, demonstrate a previously unappreciated key role for BPAG1-e in regulating keratinocyte adhesion and migration and suggest a requirement for this protein in controlling functional switching between integrin types in epithelial cells