Herlitz junctional epidermolysis bullosa (H-JEB) is an incurable, devastating, and mostly fatal inherited skin disease for which there is only supportive care. H-JEB is caused by loss-of-function mutations in ,, or , leading to complete loss of laminin 332, the major component of anchoring filaments, which mediate epidermal-dermal adherence. (laminin β3) mutations account for 80% of patients with H-JEB, and ∼95% of H-JEB-associated mutations are nonsense mutations leading to premature termination codons (PTCs). In this study, we evaluated the ability of gentamicin to induce PTC readthrough in H-JEB laminin β3-null keratinocytes transfected with expression vectors encoding eight different nonsense mutations. We found that gentamicin induced PTC readthrough in all eight nonsense mutations tested. We next used lentiviral vectors to generate stably transduced H-JEB cells with the R635X and C290X nonsense mutations. Incubation of these cell lines with various concentrations of gentamicin resulted in the synthesis and secretion of full-length laminin β3 in a dose-dependent and sustained manner. Importantly, the gentamicin-induced laminin β3 led to the restoration of laminin 332 assembly, secretion, and deposition within the dermal/epidermal junction, as well as proper polarization of α6β4 integrin in basal keratinocytes, as assessed by immunoblot analysis, immunofluorescent microscopy, and an in vitro 3D skin equivalent model. Finally, newly restored laminin 332 corrected the abnormal cellular phenotype of H-JEB cells by reversing abnormal cell morphology, poor growth potential, poor cell-substratum adhesion, and hypermotility. Therefore, gentamicin may offer a therapy for H-JEB and other inherited skin diseases caused by PTC mutations.

Extracellular heat shock protein-90alpha (eHsp90α) plays an essential role in tumour invasion and metastasis. The plasma eHsp90α levels in patients with various cancers correlate with the stages of the diseases. Nonetheless, the mechanism of action by tumour-secreted eHsp90α remained unclear. Here we show that eHsp90α accounts for approximately 1% of the total cellular Hsp90α and is associated with tumour-secreted exosomes. CRISPR-cas9 knockout of Hsp90α did not affect the overall distribution and quantity of secreted exosomes, but it caused increased exosome-associated CD9 and decreased exosome-associated TSG101, Alix, and CD63. However, Hsp90α-knockout tumour cells have not only lost their own constitutive motility, but also the ability to recruit stromal cells via secreted exosomes. These defects are specifically due to the lack of eHsp90α on tumour cell-secreted exosomes. Anti-Hsp90α antibody nullified the pro-motility activity of tumour-secreted exosomes and human recombinant Hsp90α protein fully rescued the functional defects of eHsp90α-free exosomes. Finally, while current exosome biogenesis models exclusively implicate the luminal location of host cytosolic proteins inside secreted exosomes, we provide evidence for eHsp90α location on the external surface of tumour-secreted exosomes. Taken together, this study elucidates a new mechanism of action by exosome-associated eHsp90α.

Despite years of effort and investment, there are few topical or systemic medications for skin wounds. Identifying natural drivers of wound healing could facilitate the development of new and effective treatments. When skin is injured, there is a massive increase of heat shock protein (Hsp) 90α inside the wound bed. The precise role for these Hsp90α proteins, however, was unclear. The availability of a unique mouse model that lacked the intracellular ATPase-driven chaperoning, but spared the extracellular fragment-5-supported pro-motility function of Hsp90α allowed us to test specifically the role of the non-chaperone function of Hsp90α in normal wound closure. We found that the chaperone-defective Hsp90α-Δ mutant mice showed similar wound closure rate as the wild-type Hsp90α mice. We generated recombinant proteins from the mouse cDNAs encoding the Hsp90α-Δ and wild-type Hsp90α. Topical application of Hsp90α-Δ mutant protein promoted wound closure as effectively as the full-length wild-type Hsp90α protein. More importantly, selective inhibition of the extracellular Hsp90α-Δ protein function by a monoclonal antibody targeting the fragment-5 region disrupted normal wound closure in both wild-type Hsp90α and Hsp90α-Δ mice. Thus, this study provides direct support for non-chaperone, extracellular Hsp90α as a potential driver for normal wound closure.

Generalized severe junctional epidermolysis bullosa (GS-JEB) is an incurable and fatal autosomal recessively inherited blistering skin disease caused by mutations in the LAMA3 , LAMB3 , or LAMC2 genes. Most of these mutations are nonsense mutations that create premature termination codons that lead to impaired production of functional laminin 332, a protein needed for epidermal-dermal adherence. Gentamicin induces readthrough of nonsense mutations and restores the full-length protein in various genetic diseases. Using primary keratinocytes from three GS-JEB patients, we showed that gentamicin induced functional laminin 332 that reversed a JEB-associated, abnormal cell phenotype. In a subsequent open-label trial involving the same patients, we examined whether 0.5% gentamicin ointment applied topically to open skin wounds could promote nonsense mutation readthrough and create new laminin 332 in the patients’ skin. Gentamicin-treated wounds exhibited increased expression of laminin 332 at the dermal-epidermal junction for at least 3 months and were associated with improved wound closure. There were no untoward side effects from topical gentamicin. The newly induced laminin 332 did not generate anti-laminin 332 autoantibodies in either the patients’ blood or skin. Gentamicin readthrough therapy may be a treatment for GS-JEB patients with nonsense mutations.

Periostin, an extracellular matrix macromolecule implicated in tumorigenesis, serves as a prognostic marker for many cancer types. However, there are no data on periostin expression in cutaneous squamous cell carcinoma (cSCC). This study examined periostin expression in patients with cSCC and explored its clincopathological relationship and prognosis. Using immunohistochemistry and ImageJ analysis, we compared periostin expression in 95 cSCCs across a spectrum of cSCC aggressiveness: cSCC in situ (SCCIS) (n = 25), low‐risk cSCC (LR‐cSCC) (n = 26), high‐risk cSCC (HR‐cSCC) (n = 38), and cSCC in recessive dystrophic epidermolysis bullosa patients (RDEB cSCC) (n = 6). Immunohistochemistry demonstrated periostin expression within the intra‐tumoral stroma but not within tumor cells. Periostin levels significantly (P < 0.001) increased from SCCIS, LR‐cSCC, HR‐cSCC to RDEB SCC. The stroma of most of the cSCCs we evaluated contained cancer‐associated fibroblasts with a myofibroblastic (α ‐SMA‐positive) phenotype. Co‐localization of periostin with α‐SMA, evidence of fibroblast periostin expression, and absence of keratinocyte or tumor cell periostin expression suggest that, in cSCC, periostin is a product of the peritumoral microenvironment and not the tumor cells themselves. Our data indicate that fibroblast periostin expression is highly correlated with the aggressiveness of cSCC, and may thereby provide a molecular marker that will be useful for subtyping and diagnosing cSCCs according to their biological nature.