Gasdermin A Is Required for Epidermal Cornification during Skin Barrier Regeneration and in an Atopic Dermatitis-Like Model.

Atopic dermatitis is featured with impaired skin barrier. The stratum corneum and the intercellular tight junctions constitute the permeability barrier, which is essential to protect water loss in the host and prevent pathogen entry. The epidermal barrier is constantly renewed by differentiating keratinocytes through cornification, during which autophagy contributes to elimination of organelles and nucleus. The human GSDMA and its mouse homologs Gsdma1-3 are expressed in the suprabasal epidermis. Although a pyroptotic role of GSDMA/Gsdma1 in host defense against Streptococcus pyogenes has been reported, the physiological function of Gsdma1/a2/a3 in epidermal homeostasis remains elusive. Here, through repeated epidermal barrier disruption, we found that tight junction formation and stratum corneum maturation were defective in the Gsdma1/a3-deficient epidermis. Using comparative gene profiling analysis, mitochondrial respiration measurement, and in vivo tracing of mitophagy, our data indicate that Gsdma1/a3 activation leads to mitochondrial dysfunction and subsequently facilitates mitochondrial turnover and epidermal cornification. In calcipotriol (MC903)-induced atopic dermatitis-like animal model, we showed that Gsdma1/a3-deficiency selectively enhanced the T helper type 2 response. Remarkably, the GSDMA expression is reduced in the epidermis of patients with atopic dermatitis compared with that of normal individuals. Gsdma1/a3-deficiency might be involved in atopic dermatitis pathogenesis, likely through GSDMA-mediated epidermal differentiation and cornification.

Gasdermin A3-Mediated Cell Death Causes Niche Collapse and Precocious Activation of Hair Follicle Stem Cells.

Hair follicles undergo recurrent growth, regression, and resting phases throughout postnatal life, which is supported by hair follicle stem cells. The niche components of hair follicle stem cells are important to maintain their quiescence and stemness. Gsdma3 gain-of-function mutations in mice cause chronic skin inflammation, aberrant hair cycle, and progressive hair loss, reminiscent of scarring alopecia in humans. However, the mechanism underlying these defects remains elusive. Here, we used a combined Cre/loxP and rtTA/TRE system to study the spatiotemporal effect of Gsdma3 overexpression on distinct hair cycle stages. We found that Gsdma3-mediated cell death affects anagen initiation, anagen progression, and catagen-telogen transition. Induced Gsdma3 expression causes bulge inner layer collapse and precocious hair follicle stem cell activation, leading to subsequent hair follicle degeneration. Although macrophages and dendritic cells are recruited to the bulge region, in vivo depletion of these cells using a neutralizing antibody does not alleviate cell death in the bulge or hair germ, indicating that macrophages are less likely to cause immediate hair follicle deletion. Our data suggest that dysregulated Gsdma3 causes bulge inner layer necrosis to induce club hair shedding and immediate anagen reentry without going through telogen morphology, which implicates a role for Gsdma3 in hair follicle stem cell niche maintenance.

Hes1 regulates anagen initiation and hair follicle regeneration through modulation of hedgehog signaling.

Adult hair follicles undergo repeated cycling of regression (catagen), resting (telogen), and growth (anagen), which is maintained by hair follicle stem cells (HFSCs). The mechanism underlying hair growth initiation and HFSC maintenance is not fully understood. Here, by epithelial deletion of Hes1, a major Notch downstream transcriptional repressor, we found that hair growth is retarded, but the hair cycle progresses normally. Hes1 is specifically upregulated in the lower bulge/HG during anagen initiation. Accordingly, loss of Hes1 results in delayed activation of the secondary hair germ (HG) and shortened anagen phase. This developmental delay causes reduced hair shaft length but not identity changes in follicular lineages. Remarkably, Hes1 ablation results in impaired hair regeneration upon repetitive depilation. Microarray gene profiling on HFSCs indicates that Hes1 modulates Shh responsiveness in anagen initiation. Using primary keratinocyte cultures, we demonstrated that Hes1 deletion negatively influences ciliogenesis and Smoothened ciliary accumulation upon Shh treatment. Furthermore, transient application of Smoothened agonist during repetitive depilation can rescue anagen initiation and HFSC self-renewal in Hes1-deficient hair follicles. We reveal a critical function of Hes1 in potentiating Shh signaling in anagen initiation, which allows sufficient signaling strength to expand the HG and replenish HFSCs to maintain the hair cycle homeostasis.

Protein Palmitoylation by ZDHHC13 Protects Skin against Microbial-Driven Dermatitis.

Atopic dermatitis is a complex chronic inflammatory skin disorder that results from intimate interactions among genetic predisposition, host environment, skin barrier defects, and immunological factors. However, a clear genetic roadmap leading to atopic dermatitis remains to be fully explored. From a genome-wide mutagenesis screen, deficiency of ZDHHC13, a palmitoylacyl transferase, has previously been associated with skin and multitissue inflammatory phenotypes. Here, we report that ZDHHC13 is required for skin barrier integrity and that deficiency of ZDHHC13 renders mice susceptible to environmental bacteria, resulting in persistent skin inflammation and an atopic dermatitis-like disease. This phenotype is ameliorated in a germ-free environment and is also attenuated by antibiotic treatment, but not by deletion of the Rag1 gene, suggesting that a microbial factor triggers inflammation rather than intrinsic adaptive immunity. Furthermore, skin from ZDHHC13-deficient mice has both elevated levels of IL-33 and type 2 innate lymphoid cells, reinforcing the role of innate immunity in the development of atopic dermatitis. In summary, our study suggests that loss of ZDHHC13 in skin impairs the integrity of multiple barrier functions and leads to a dermatitis lesion in response to microbial encounters.

N-terminal functional domain of Gasdermin A3 regulates mitochondrial homeostasis via mitochondrial targeting.

BACKGROUND: The epidermis forms a critical barrier that is maintained by orchestrated programs of proliferation, differentiation, and cell death. Gene mutations that disturb this turnover process may cause skin diseases. Human GASDERMIN A (GSDMA) is frequently silenced in gastric cancer cell lines and its overexpression has been reported to induce apoptosis. GSDMA has also been linked with airway hyperresponsiveness in genetic association studies. The function of GSDMA in the skin was deduced by dominant mutations in mouse gasdermin A3 (Gsdma3), which caused skin inflammation and hair loss. However, the mechanism for the autosomal dominance of Gsdma3 mutations and the mode of Gsdma3's action remain unanswered. RESULTS: We demonstrated a novel function of Gsdma3 in modulating mitochondrial oxidative stress. We showed that Gsdma3 is regulated by intramolecular fold-back inhibition, which is disrupted by dominant mutations in the C-terminal domain. The unmasked N-terminal domain of Gsdma3 associates with Hsp90 and is delivered to mitochondrial via mitochondrial importer receptor Tom70, where it interacts with the mitochondrial chaperone Trap1 and causes increased production of mitochondrial reactive oxygen species (ROS), dissipation of mitochondrial membrane potential, and mitochondrial permeability transition (MPT). Overexpression of the C-terminal domain of Gsdma3 as well as pharmacological interventions of mitochondrial translocation, ROS production, and MPT pore opening alleviate the cell death induced by Gsdma3 mutants. CONCLUSIONS: Our results indicate that the genetic mutations in the C-terminal domain of Gsdma3 are gain-of-function mutations which unmask the N-terminal functional domain of Gsdma3. Gsdma3 regulates mitochondrial oxidative stress through mitochondrial targeting. Since mitochondrial ROS has been shown to promote epidermal differentiation, we hypothesize that Gsdma3 regulates context-dependent response of keratinocytes to differentiation and cell death signals by impinging on mitochondria.

Divergent signaling pathways cooperatively regulate TGFß induction of cysteine-rich protein 2 in vascular smooth muscle cells.

BACKGROUND: Vascular smooth muscle cells (VSMCs) of the arterial wall play a critical role in the development of occlusive vascular diseases. Cysteine-rich protein 2 (CRP2) is a VSMC-expressed LIM-only protein, which functionally limits VSMC migration and protects against pathological vascular remodeling. The multifunctional cytokine TGFß has been implicated to play a role in the pathogenesis of atherosclerosis through numerous downstream signaling pathways. We showed previously that TGFß upregulates CRP2 expression; however, the detailed signaling mechanisms remain unclear. RESULTS: TGFß treatment of VSMCs activated both Smad2/3 and ATF2 phosphorylation. Individually knocking down Smad2/3 or ATF2 pathways with siRNA impaired the TGFß induction of CRP2, indicating that both contribute to CRP2 expression. Inhibiting TßRI kinase activity by SB431542 or TßRI knockdown abolished Smad2/3 phosphorylation but did not alter ATF2 phosphorylation, indicating while Smad2/3 phosphorylation was TßRI-dependent ATF2 phosphorylation was independent of TßRI. Inhibiting Src kinase activity by SU6656 suppressed TGFß-induced RhoA and ATF2 activation but not Smad2 phosphorylation. Blocking ROCK activity, the major downstream target of RhoA, abolished ATF2 phosphorylation and CRP2 induction but not Smad2 phosphorylation. Furthermore, JNK inhibition with SP600125 reduced TGFß-induced ATF2 (but not Smad2) phosphorylation and CRP2 protein expression while ROCK inhibition blocked JNK activation. These results indicate that downstream of TßRII, Src family kinase-RhoA-ROCK-JNK signaling pathway mediates TßRI-independent ATF2 activation. Promoter analysis revealed that the TGFß induction of CRP2 was mediated through the CRE and SBE promoter elements that were located in close proximity. CONCLUSIONS: Our results demonstrate that two signaling pathways downstream of TGFß converge on the CRE and SBE sites of the Csrp2 promoter to cooperatively control CRP2 induction in VSMCs, which represents a previously unrecognized mechanism of VSMC gene induction by TGFß.

Differential response of epithelial stem cell populations in hair follicles to TGF-ß signaling.

Epidermal stem cells residing in different locations in the skin continuously self-renew and differentiate into distinct cell lineages to maintain skin homeostasis during postnatal life. Murine epidermal stem cells located at the bulge region are responsible for replenishing the hair lineage, while the stem cells at the isthmus regenerate interfollicular epidermis and sebaceous glands. In vitro cell culture and in vivo animal studies have implicated TGF-ß signaling in the maintenance of epidermal and hair cycle homeostasis. Here, we employed a triple transgenic animal model that utilizes a combined Cre/loxP and rtTA/TRE system to allow inducible and reversible inhibition of TGF-ß signaling in hair follicle lineages and suprabasal layer of the epidermis. Using this animal model, we have analyzed the role of TGF-ß signaling in distinct phases of the hair cycle. Transient abrogation of TGF-ß signaling does not prevent catagen progression; however, it induces aberrant proliferation and differentiation of isthmus stem cells to epidermis and sebocyte lineages and a blockade in anagen re-entry as well as results in an incomplete hair shaft development. Moreover, ablation of TGF-ß signaling during anagen leads to increased apoptosis in the secondary hair germ and bulb matrix cells. Blocking of TGF-ß signaling in bulge stem cell cultures abolishes their colony-forming ability, suggesting that TGF-ß signaling is required for the maintenance of bulge stem cells. At the molecular level, inhibition of TGF-ß signaling results in a decrease in both Lrig1-expressing isthmus stem cells and Lrg5-expressing bulge stem cells, which may account for the phenotypes seen in our animal model. These data strongly suggest that TGF-ß signaling plays an important role in regulating the proliferation, differentiation, and apoptosis of distinct epithelial stem cell populations in hair follicles.

Notch signaling prevents mucous metaplasia in mouse conducting airways during postnatal development.

Goblet cell metaplasia and mucus overproduction contribute to the pathogenesis of chronic lung diseases, including asthma and chronic obstructive pulmonary disease (COPD). Notch signaling regulates cell fate decisions and is crucial in controlling goblet cell differentiation in the gut epithelium. Little is known, however, about how endogenous Notch signaling influences the goblet cell differentiation program that takes place in the postnatal lung. Using a combination of genetic and in vitro approaches here we provide evidence of a novel role for Notch in restricting goblet cell differentiation in the airway epithelium during the postnatal period. Conditional inactivation of the essential Notch pathway component Pofut1 (protein O-fucosyltransferase1) in Tgfb3-Cre-expressing mice resulted in an aberrant postnatal airway phenotype characterized by marked goblet cell metaplasia, decreased Clara cell number and increase in ciliated cells. The presence of the same phenotype in mice in which the Notch transcriptional effector Rbpjk was deleted indicated the involvement of the canonical Notch pathway. Lineage study in vivo suggested that goblet cells originated from a subpopulation of Clara cells largely present in proximal airways in which Notch was disrupted. The phenotype was confirmed by a panel of goblet cell markers, showed no changes in cell proliferation or altered expression of proinflammatory cytokines and was associated with significant downregulation of the bHLH transcriptional repressor Hes5. Luciferase reporter analysis suggested that Notch directly repressed MUC5AC transcription in lung epithelial cells. The data suggested that during postnatal life Notch is required to prevent Clara cells from differentiating into goblet cells.

Generation of mice with a conditional allele for the p75(NTR) neurotrophin receptor gene.

The p75(NTR) neurotrophin receptor has been implicated in multiple biological and pathological processes. While significant advances have recently been made in understanding the physiologic role of p75(NTR) , many details and aspects remain to be determined. This is in part because the two existing knockout mouse models (Exons 3 or 4 deleted, respectively), both display features that defy definitive conclusions. Here we describe the generation of mice that carry a conditional p75(NTR) (p75(NTR-FX) ) allele made by flanking Exons 4-6, which encode the transmembrane and all cytoplasmic domains, by loxP sites. To validate this novel conditional allele, both neural crest-specific p75(NTR) /Wnt1-Cre mutants and conventional p75(NTR) null mutants were generated. Both mutants displayed abnormal hind limb reflexes, implying that loss of p75(NTR) in neural crest-derived cells causes a peripheral neuropathy similar to that seen in conventional p75(NTR) mutants. This novel conditional p75(NTR) allele will offer new opportunities to investigate the role of p75(NTR) in specific tissues and cells.

Notch signaling regulates late-stage epidermal differentiation and maintains postnatal hair cycle homeostasis.

BACKGROUND: Notch signaling involves ligand-receptor interactions through direct cell-cell contact. Multiple Notch receptors and ligands are expressed in the epidermis and hair follicles during embryonic development and the adult stage. Although Notch signaling plays an important role in regulating differentiation of the epidermis and hair follicles, it remains unclear how Notch signaling participates in late-stage epidermal differentiation and postnatal hair cycle homeostasis. METHODOLOGY AND PRINCIPAL FINDINGS: We applied Cre/loxP system to generate conditional gene targeted mice that allow inactivation of critical components of Notch signaling pathway in the skin. Rbpj, the core component of all four Notch receptors, and Pofut1, an essential factor for ligand-receptor interactions, were inactivated in hair follicle lineages and suprabasal layer of the epidermis using the Tgfb3-Cre mouse line. Rbpj conditional inactivation resulted in granular parakeratosis and reactive epidermal hyperplasia. Pofut1 conditional inactivation led to ultrastructural abnormalities in the granular layer and altered filaggrin processing in the epidermis, suggesting a perturbation of the granular layer differentiation. Disruption of Pofut1 in hair follicle lineages resulted in aberrant telogen morphology, a decrease of bulge stem cell markers, and a concomitant increase of K14-positive keratinocytes in the isthmus of mutant hair follicles. Pofut1-deficent hair follicles displayed a delay in anagen re-entry and dysregulation of proliferation and apoptosis during the hair cycle transition. Moreover, increased DNA double stand breaks were detected in Pofut1-deficent hair follicles, and real time PCR analyses on bulge keratinocytes isolated by FACS revealed an induction of DNA damage response and a paucity of DNA repair machinery in mutant bulge keratinocytes. SIGNIFICANCE: our data reveal a role for Notch signaling in regulating late-stage epidermal differentiation. Notch signaling is required for postnatal hair cycle homeostasis by maintaining proper proliferation and differentiation of hair follicle stem cells.

Tissue-specific expression of Cre recombinase from the Tgfb3 locus.

Tgfb3, a member of the TGF-beta superfamily, is tightly regulated, both spatially and temporally, during embryogenesis. Previous mouse knockout studies have demonstrated that Tgfb3 is absolutely required for normal palatal fusion and pulmonary development. We have generated a novel tool to ablate genes in Tgfb3-expressing cells by targeting the promoterless Cre-pgk-Neo cassette into exon 1 of the mouse Tgfb3 gene, which generates a functionally null Tgfb3 allele. Using the Rosa26 reporter assay, we demonstrate that Cre-induced recombination was already induced at embryonal day 10 (E10) in the ventricular myocardium, limb buds, and otic vesicles. At E14, robust recombination was detected in the prefusion palatal epithelium. Deletion of the TGF-beta type I receptor Alk5 (Tgfbr1) specifically in Tgfb3 expressing cells using the Tgfb3-Cre driver line lead to a cleft palate phenotype similar to that seen in conventional Tgfb3 null mutants. In addition, Alk5/ Tgfb3-Cre mice displayed hydrocephalus, and severe intracranial bleeding due to germinal matrix hemorrhage.

Tgfb1 expressed in the Tgfb3 locus partially rescues the cleft palate phenotype of Tgfb3 null mutants.

Although TGF-beta isoforms (TGF-beta1-3) display very similar biochemical characteristics in vitro, it has been determined that they demonstrate different or even opposing effects in vivo. During embryogenesis, TGF-betas play important roles in several developmental processes. Tgfb3 is strongly expressed in the prefusion palatal epithelium, and mice lacking Tgfb3 display a cleft of the secondary palate. To test whether the effect of TGF-beta3 in palatogenesis is isoform-specific in vivo, we generated a knockin mouse by replacing the coding region of exon1 in the Tgfb3 gene with the full-length Tgfb1 cDNA, which resulted in the expression of Tgfb1 in the Tgfb3 expressing domain. The homozygote knockin mice display a complete fusion at the mid-portion of the secondary palate, while the most anterior and posterior regions fail to fuse appropriately indicating that in vivo replacement of TGF-beta3 with TGF-beta1 can only partially correct the epithelial fusion defect of Tgfb3 knockout embryos. Palatal shelves of Tgfb1 knockin homozygote mice adhere, intercalate, and form characteristic epithelial triangles. However, decreased apoptosis in the midline epithelium, slower breakdown of the basement membrane and a general delay in epithelial fusion were observed when compared to control littermates. These results demonstrate an isoform-specific role for TGF-beta3 in the palatal epithelium during palate formation, which cannot be fully substituted with TGF-beta1.

The divergent DSL ligand Dll3 does not activate Notch signaling but cell autonomously attenuates signaling induced by other DSL ligands.

Mutations in the DSL (Delta, Serrate, Lag2) Notch (N) ligand Delta-like (Dll) 3 cause skeletal abnormalities in spondylocostal dysostosis, which is consistent with a critical role for N signaling during somitogenesis. Understanding how Dll3 functions is complicated by reports that DSL ligands both activate and inhibit N signaling. In contrast to other DSL ligands, we show that Dll3 does not activate N signaling in multiple assays. Consistent with these findings, Dll3 does not bind to cells expressing any of the four N receptors, and N1 does not bind Dll3-expressing cells. However, in a cell-autonomous manner, Dll3 suppressed N signaling, as was found for other DSL ligands. Therefore, Dll3 functions not as an activator as previously reported but rather as a dedicated inhibitor of N signaling. As an N antagonist, Dll3 promoted Xenopus laevis neurogenesis and inhibited glial differentiation of mouse neural progenitors. Finally, together with the modulator lunatic fringe, Dll3 altered N signaling levels that were induced by other DSL ligands.

Fringe glycosyltransferases differentially modulate Notch1 proteolysis induced by Delta1 and Jagged1.

Fringe O-fucose-beta1,3-N-acetylglucosaminyltransferases modulate Notch signaling by potentiating signaling induced by Delta-like ligands, while inhibiting signaling induced by Serrate/Jagged1 ligands. Based on binding studies, the differential effects of Drosophila fringe (DFng) on Notch signaling are thought to result from alterations in Notch glycosylation that enhance binding of Delta to Notch but reduce Serrate binding. Here, we report that expression of mammalian fringe proteins (Lunatic [LFng], Manic [MFng], or Radical [RFng] Fringe) increased Delta1 binding and activation of Notch1 signaling in 293T and NIH 3T3 cells. Although Jagged1-induced signaling was suppressed by LFng and MFng, RFng enhanced signaling induced by either Delta1 or Jagged1, underscoring the diversity of mammalian fringe glycosyltransferases in regulating signaling downstream of different ligand-receptor combinations. Interestingly, suppression of Jagged1-induced Notch1 signaling did not correlate with changes in Jagged1 binding as found for Delta1. Our data support the idea that fringe glycosylation increases Delta1 binding to potentiate signaling, but we propose that although fringe glycosylation does not reduce Jagged1 binding to Notch1, the resultant ligand-receptor interactions do not effectively promote Notch1 proteolysis required for activation of downstream signaling events.

c-SRC mediates neurite outgrowth through recruitment of Crk to the scaffolding protein Sin/Efs without altering the kinetics of ERK activation.

SRC family kinases have been consistently and recurrently implicated in neurite extension events, yet the mechanism underlying their neuritogenic role has remained elusive. We report that epidermal growth factor (EGF) can be converted from a non-neuritogenic into a neuritogenic factor through moderate activation of endogenous SRC by receptor-protein-tyrosine phosphatase alpha (a physiological SRC activator). We show that such a qualitative change in the response to EGF is not accompanied by changes in the extent or kinetics of ERK induction in response to this factor. Instead, the pathway involved relies on increased tyrosine phosphorylation of, and recruitment of Crk to, the SRC substrate Sin/Efs. The latter is a scaffolding protein structurally similar to the SRC substrate Cas, tyrosine phosphorylation of which is critical for migration in fibroblasts and epithelial cells. Expression of a dominant negative version of Sin interfered with receptor-protein-tyrosine phosphatase alpha/EGF- as well as fibroblast growth factor-induced neurite outgrowth. These observations uncouple neuritogenic signaling in PC12 cells from sustained activation of ERK kinases and for the first time identify an effector of SRC function in neurite extension.