An unanticipated discourse of HB-EGF with VANGL2 signaling during embryo implantation.

Implantation is the first direct encounter between the embryo and uterus during pregnancy, and Hbegf is the earliest known molecular signaling for embryo-uterine crosstalk during implantation. The downstream effectors of heparin-binding EGF (HB-EGF) in implantation remain elusive due to the complexity of EGF receptor family. This study shows that the formation of implantation chamber (crypt) triggered by HB-EGF is disrupted by uterine deletion of Vangl2, a key planar cell polarity component (PCP). We found that HB-EGF binds to ERBB2 and ERBB3 to recruit VANGL2 for tyrosine phosphorylation. Using in vivo models, we show that uterine VAGL2 tyrosine phosphorylation is suppressed in Erbb2/Erbb3 double conditional knockout mice. In this context, severe implantation defects in these mice lend support to the critical role of HB-EGF-ERBB2/3-VANGL2 in establishing a two-way dialogue between the blastocyst and uterus. In addition, the result addresses an outstanding question how VANGL2 is activated during implantation. Taken together, these observations reveal that HB-EGF regulates the implantation process by influencing uterine epithelial cell polarity comprising VANGL2.

EGFR ligands synergistically increase IL-17A-induced expression of psoriasis signature genes in human keratinocytes via IκBζ and Bcl3.

Various epidermal growth factor receptor (EGFR) ligands are highly expressed in the epidermis of psoriasis lesions, and abnormal EGFR activation appears to be involved in the pathogenesis of psoriasis. However, how EGFR signaling contributes to the development of psoriasis is unclear. Interleukin (IL)-17A, a critical effector of the IL-23/IL-17A pathway, increases the expression of psoriasis signature genes in keratinocytes and plays an essential role in the pathogenesis of psoriasis by inducing IκBζ, a critical transcriptional regulator in psoriasis. In this study, we stimulated primary human keratinocytes with IL-17A and various EGFR ligands to investigate whether EGFR ligands regulate the expression of psoriasis signature genes. In cultured normal human keratinocytes and a living skin equivalent, EGFR ligands did not induce psoriasis-related gene expression, but significantly enhanced the IL-17A-mediated induction of various psoriasis signature genes, including antimicrobial peptides, cytokines, and chemokines. This was dependent on an EGFR activation-mediated synergistic increase in IL-17A-induced IκBζ expression and was partially mediated by the EGFR-dependent upregulation of Bcl3. Therefore, EGFR ligands can act as synergistic agents of IL-17A signaling by stimulating the epidermal production of psoriasis signature genes in psoriasis lesions. This study reveals a potential mechanism by which EGFR signaling contributes to the pathogenesis of psoriasis.

Egr3 as an important regulator of uterine decidualization through targeting Hand2.

Early growth response 3 (Egr3) is required for embryogenesis, but little understanding is usable about its function in embryo implantation and decidualization. The present study exhibited an obvious localization of Egr3 in luminal epithelium and subluminal stroma at implantation sites. Administration of estrogen brought about a distinct gather of Egr3 mRNA in uterine luminal and glandular epithelia. Meanwhile, Egr3 was visualized in the decidua where it might facilitate the proliferation of stromal cells via Ccnd3 and accelerate stromal differentiation, testifying the significance of Egr3 in decidualization. In ovariectomized mice uteri or stromal cells, progesterone advanced the expression of Egr3 whose obstruction counteracted the inducement of stromal differentiation by progesterone. Consistently, Egr3 mediated the influence of cAMP and heparin-binding EGF-like growth factor (HB-EGF) on the differentiation program. Additionally, cAMP-protein kinase A (PKA) signaling mediated the adjustment of progesterone on Egr3. Impediment of HB-EGF antagonized the ascendance of Egr3 conferred by cAMP. In stromal cells, Egr3 activated the transcription of Hand2 whose promoter region exhibited the binding enrichment of Egr3. Activation of Hand2 relieved the weakness of stromal differentiation by Egr3 hinderance, whereas knockdown of Hand2 neutralized the guidance of Egr3 overexpression on the differentiation program. Collectively, Egr3 was identified as an important regulator of uterine decidualization through targeting Hand2 in response to progesterone/cAMP/HB-EGF pathway.

Effect of pituitary adenylate cyclase-activating polypeptide supplementation, applied during or after vitrification on mouse embryo.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide with widespread occurrence and diverse functions. It occurs in high levels in the gonads suggesting a potential central role in reproduction. The aim of our study was to assess the effect of PACAP treatment during embryo vitrification on the developmental rate and the expression of the heparin-binding EGF-like growth factor gene (Hbegf). Mouse embryos, obtained from superovulated females were allocated into the four treatment groups. In EM1 and EM2, the embryos were prepared for vitrification in an Equilibration Solution that was supplemented with 1 or 2 µM PACAP1-38, respectively. The embyos in groups CM1 and CM2 were not treated prior to vitrification but were cultured in a medium supplemented with 1 or 2 µM PACAP1-38 after thawing. The Vitrified Control group consisted of embryos vitrified and thawed then cultured without PACAP1-38 treatment. A non-vitrified, non-treated Fresh Control group was also used. After 24 h of culture, the developmental rate of the embryos, as well as the relative expression level of the Hbegf gene, as determined by qPCR, were compared among groups. Higher developmental rate and Hbegf gene expression level were found in the embryos treated with a higher concentration of PACAP. These results indicate that PACAP treatment has a beneficial effect on the survival and development of vitrified/thawed mouse embryos.

Small GTP-binding protein Rap1 mediates EGF and HB-EGF signaling and modulates EGF receptor expression in HTR-8/SVneo extravillous trophoblast cells.

Purpose: Extravillous trophoblasts (EVTs) invade the endometrium to establish a fetomaternal interaction during pregnancy. Epidermal growth factor (EGF) and heparin-binding EGF-like growth factor (HB-EGF) stimulate EVT invasion by binding to the EGF receptor (EGFR). We examined the role of the small GTP-binding protein Rap1 in EGF- and HB-EGF-stimulated EVT invasion. Methods: Expression of Rap1 in the first-trimester placenta was examined by immunohistochemistry. Effect of EGF or HB-EGF on Rap1 activation (GTP-Rap1) and Rap1 knockdown on invasion was assessed in EVT cell line (HTR-8/SVneo). In addition, effect of Rap1 knockdown and Rap1GAP (a Rap1 inactivator) overexpression on the activation of EGF signaling and EGFR expression were examined. Results: Rap1 was expressed by EVTs, villous cytotrophoblasts, and syncytiotrophoblasts in the placenta. EGF and HB-EGF activated Rap1 and promoted invasion of HTR-8/SVneo, and these effects were inhibited by Rap1 knockdown. The EGF- and HB-EGF-induced phosphorylation of AKT, ERK1/2, p38MAPK, and Src was inhibited by Rap1 knockdown. Furthermore, the knockdown of Rap1 reduced the EGFR protein level. Overexpression of Rap1GAP repressed EGF- and HB-EGF-induced Rap1 activation and reduced EGFR expression. Conclusion: Rap1 may function as a mediator of EGF and HB-EGF signaling pathways and can modulate EGFR expression in EVTs during placental development.

Elevated HB-EGF expression in neural stem cells causes middle age obesity by suppressing Hypocretin/Orexin expression.

Obesity is common in the middle aged population and it increases the risks of diabetes, cardiovascular diseases, certain cancers, and dementia. Yet, its etiology remains incompletely understood. Here, we show that ectopic expression of HB-EGF, an important regulator of neurogenesis, in Nestin+ neuroepithelial progenitors with the Cre-LoxP system leads to development of spontaneous middle age obesity in male mice accompanied by hyperglycemia and insulin resistance. The Nestin-HB-EGF mice show decreases in food uptake, energy expenditure, and physical activity, suggesting that reduced energy expenditure underlies the pathogenesis of this obesity model. However, HB-EGF expression in appetite-controlling POMC or AgRP neurons or adipocytes fails to induce obesity. Mechanistically, HB-EGF suppresses expression of Hypocretin/Orexin, an orexigenic neuropeptide hormone, in the hypothalamus of middle aged Nestin-HB-EGF mice. Hypothalamus Orexin administration alleviates the obese and hyperglycemic phenotypes in Nestin-HB-EGF mice. This study uncovers an important role for HB-EGF in regulating Orexin expression and energy expenditure and establishes a midlife obesity model whose pathogenesis involves age-dependent changes in hypothalamus neurons.

HB-EGF upregulates StAR expression and stimulates progesterone production through ERK1/2 signaling in human granulosa-lutein cells.

BACKGROUND: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) belongs to the epidermal growth factor (EGF) family of growth factors. HB-EGF and its receptors, epidermal growth factor receptor (EGFR) and HER4, are expressed in the human corpus luteum. HB-EGF has been shown to regulate luteal function by preventing cell apoptosis. Steroidogenesis is the primary function of the human corpus luteum. Steroidogenic acute regulatory protein (StAR) plays a critical role in steroidogenesis. StAR expression and progesterone (P4) production in human granulosa-lutein (hGL) cells have been shown to be upregulated by a ligand of EGFR, amphiregulin. However, whether HB-EGF can achieve the same effects remains unknown. METHODS: A steroidogenic human ovarian granulosa-like tumor cell line, KGN, and primary culture of hGL cells obtained from patients undergoing in vitro fertilization treatment were used as experimental models. The underlying molecular mechanisms mediating the effects of HB-EGF on StAR expression and P4 production were explored by a series of in vitro experiments. RESULTS: Western blot showed that EGFR, HER2, and HER4 were expressed in both KGN and hGL cells. Treatment with HB-EGF for 24 h induced StAR expression but did not affect the expression of steroidogenesis-related enzymes, P450 side chain cleavage enzyme, 3ß-hydroxysteroid dehydrogenase, and aromatase. Using pharmacological inhibitors and a siRNA-mediated knockdown approach, we showed that EGFR, HER4, but not HER2, were required for HB-EGF-stimulated StAR expression and P4 production. In addition, HB-EGF-induced upregulations of StAR expression and P4 production were mediated by the activation of the ERK1/2 signaling pathway. CONCLUSION: This study increases the understanding of the physiological role of HB-EGF in human luteal functions. Video Abstract.

The matricellular protein decorin delivered intradermally with coacervate improves wound resolution in the CXCR3-deficient mouse model of hypertrophic scarring.

Cutaneous wound healing is an intricate orchestration of three overlapping phases of repair that encompass numerous cell types, signalling cascades, and microenvironment modifications to reach a successful resolution. Disruption of any of these steps will create an abnormal healing response resulting in either ulceration or excessive scarring. It has become evident that the extracellular matrix and its associated components are key orchestrators during this process. One of these essential matrix proteins is decorin, a small leucine-rich proteoglycan (SLRP) that acts as a regulator of collagen fibrillogenesis and a non-competitive inhibitor of multiple growth factors signalling cascades. Decorin is a necessary shut-off switch for the pro-reparative mechanism of the tissue replacement phase and limits the occurrence of hypertrophic scarring by preventing excessive repair. We investigated the use of decorin as a therapeutic by administering the matrix protein anchored in a slow-release coacervate in a hypertrophic scarring mouse model. The results show that early wound healing phase measurements exhibit little difference in performance compared to our coacervate-only baseline or HB-EGF-treated control mice. However, during the resolution phase of wound healing, the decorin-treatment significantly reduces cutaneous thickness, enhances collagen alignment, and improves overall wound scoring in the mice. Thus, mice treated with decorin display better healing outcomes and could limit the hypertrophic scarring phenotype in the coacervate only, and HB-EGF controls. These results suggest that decorin may be a promising tool and alternative therapy for patients who suffer from over-exuberant matrix deposition during wound healing.

Matrix Metalloproteinase-10 in Kidney Injury Repair and Disease.

Matrix metalloproteinase-10 (MMP-10) is a zinc-dependent endopeptidase with the ability to degrade a broad spectrum of extracellular matrices and other protein substrates. The expression of MMP-10 is induced in acute kidney injury (AKI) and chronic kidney disease (CKD), as well as in renal cell carcinoma (RCC). During the different stages of kidney injury, MMP-10 may exert distinct functions by cleaving various bioactive substrates including heparin-binding epidermal growth factor (HB-EGF), zonula occludens-1 (ZO-1), and pro-MMP-1, -7, -8, -9, -10, -13. Functionally, MMP-10 is reno-protective in AKI by promoting HB-EGF-mediated tubular repair and regeneration, whereas it aggravates podocyte dysfunction and proteinuria by disrupting glomerular filtration integrity via degrading ZO-1. MMP-10 is also involved in cancerous invasion and emerges as a promising therapeutic target in patients with RCC. As a secreted protein, MMP-10 could be detected in the circulation and presents an inverse correlation with renal function. Due to the structural similarities between MMP-10 and the other MMPs, development of specific inhibitors targeting MMP-10 is challenging. In this review, we summarize our current understanding of the role of MMP-10 in kidney diseases and discuss the potential mechanisms of its actions.

Malic enzyme 1 is important for uterine decidualization in response to progesterone/cAMP/PKA/HB-EGF pathway.

Malic enzyme 1 (Me1), a member of the malic enzymes involving in glycolytic pathway and citric acid cycle, is essential for the energy metabolism and maintenance of intracellular redox balance state, but its physiological role and regulatory mechanism in the uterine decidualization are still unknown. Current study showed that Me1 was strongly expressed in decidual cells, and could promote the proliferation and differentiation of stromal cells followed by an accelerated cell cycle transition, indicating an importance of Me1 in the uterine decidualization. Silencing of Me1 attenuated NADPH generation and reduced GR activity, while addition of NADPH improved the defect of GR activity elicited by Me1 depletion. Further analysis found that Me1 modulated intracellular GSH content via GR. Meanwhile, Me1 played a role in maintaining mitochondrial function as indicated by these observations that blockadge of Me1 led to the accumulation of mitochondrial O2- level and decreased ATP production and mtDNA copy numbers accompanied with defective mitochondrial membrane potential. In uterine stromal cells, progesterone induced Me1 expression through PR-cAMP-PKA pathway. Knockdown of HB-EGF might impede the regulation of progesterone and cAMP on Me1. Collectively, Me1 is essential for uterine decidualization in response to progesterone/cAMP/PKA/HB-EGF pathway and plays an important role in preventing mitochondrial dysfunction.

HB-EGF-induced IL-8 secretion from airway epithelium leads to lung fibroblast proliferation and migration.

BACKGROUND: We have reported that heparin-binding epidermal growth factor (HB-EGF) is increased in patients with chronic obstructive pulmonary disease (COPD) and associated with collagen deposition, but the mechanisms remain unclear. In the present study, we aimed to investigated the inflammatory cytokines secreted by bronchial epithelial cells following exposure to HB-EGF that promoted proliferation and migration of human lung fibroblast. METHODS: HB-EGF-induced inflammatory cytokines were assayed in two airway epithelial cells (primary human bronchial epithelial cells [HBECs] and BEAS-2B cells). Moreover, the culture supernatants derived from HB-EGF-treated HBECs and BEAS-2B cells were added to human primary lung fibroblasts. The effect of culture supernatants on proliferation and migration of fibroblasts was assessed. RESULTS: IL-8 expression was significantly increased in bronchial epithelial cells treated with HB-EGF, which was at least partially dependent on NF-kB pathways activation. HB-EGF-induced IL-8 was found to further promote lung fibroblasts proliferation and migration, and the effects were attenuated after neutralizing IL-8. CONCLUSIONS: These findings suggest that HB-EGF may be involved in the pathology of airway fibrosis by induction of IL-8 from airway epithelium, subsequently causing lung fibroblasts proliferation and migration. Thus, inhibition of HBEGF and/or IL-8 production could prevent the development of airway fibrosis by modulating fibroblast activation.

Aloysia Citrodora Essential Oil Inhibits Melanoma Cell Growth and Migration by Targeting HB-EGF-EGFR Signaling.

Patients diagnosed with melanoma have a poor prognosis due to regional invasion and metastases. The receptor tyrosine kinase epidermal growth factor receptor (EGFR) is found in a subtype of melanoma with a poor prognosis and contributes to drug resistance. Aloysia citrodora essential oil (ALOC-EO) possesses an antitumor effect. Understanding signaling pathways that contribute to the antitumor of ALOC-EO is important to identify novel tumor types that can be targeted by ALOC-EO. Here, we investigated the effects of ALOC-EO on melanoma growth and tumor cell migration. ALOC-EO blocked melanoma growth in vitro and impaired primary tumor cell growth in vivo. Mechanistically, ALOC-EO blocked heparin-binding-epidermal growth factor (HB-EGF)-induced EGFR signaling and suppressed ERK1/2 phosphorylation. Myelosuppressive drugs upregulated HB-EGF and EGFR expression in melanoma cells. Cotreatment of myelosuppressive drugs with ALOC-EO improved the antitumor activity and inhibited the expression of matrix metalloproteinase-7 and -9 and a disintegrin and metalloproteinase domain-containing protein9. In summary, our study demonstrates that ALOC-EO blocks EGFR and ERK1/2 signaling, with preclinical efficacy as a monotherapy or in combination with myelosuppressive drugs in melanoma.

Polarization of ADAM17-driven EGFR signalling in electric field-guided collective migration of epidermal sheets.

Endogenous electric field is considered to play an important role in promoting collective migration of epidermis to the wound centre. However, most studies are focused on the effect of bioelectric field on the movement and migration of single epithelial cell; the molecular mechanisms about collective migration of epidermal monolayers remain unclear. Here, we found that EFs dramatically promoted the collective migration of HaCaT cells towards the anode, activated the sheddase activity of ADAM17 and increased the phosphorylation level of EGFR. Moreover, EGFR phosphorylation and HB-EGF shedding level were significantly decreased by the ADAM17 inhibitor TAPI-2 or siADAM17 under EFs, which subsequently attenuated the directed migration of HaCaT sheets. Notably, the inhibition of EF-regulated collective migration by siADAM17 was rescued by addition of recombinant HB-EGF. Furthermore, we observed that F-actin was dynamically polarized along the leading edge of the migrated sheets under EFs and that this polarization was regulated by ADAM17/HB-EGF/EGFR signalling. In conclusion, our study indicated that ADAM17 contributed to the collective directional movement of the epidermal monolayer by driving HB-EGF release and activating EGFR under EFs, and this pathway also mediated the polarization of F-actin in migrating sheets, which is essential in directional migration.

Endothelial Cells Regulate Physiological Cardiomyocyte Growth via VEGFR2-Mediated Paracrine Signaling.

BACKGROUND: Heart failure, which is a major global health problem, is often preceded by pathological cardiac hypertrophy. The expansion of the cardiac vasculature, to maintain adequate supply of oxygen and nutrients, is a key determinant of whether the heart grows in a physiological compensated manner or a pathological decompensated manner. Bidirectional endothelial cell (EC)-cardiomyocyte (CMC) cross talk via cardiokine and angiocrine signaling plays an essential role in the regulation of cardiac growth and homeostasis. Currently, the mechanisms involved in the EC-CMC interaction are not fully understood, and very little is known about the EC-derived signals involved. Understanding how an excess of angiogenesis induces cardiac hypertrophy and how ECs regulate CMC homeostasis could provide novel therapeutic targets for heart failure. METHODS: Genetic mouse models were used to delete vascular endothelial growth factor (VEGF) receptors, adeno-associated viral vectors to transduce the myocardium, and pharmacological inhibitors to block VEGF and ErbB signaling in vivo. Cell culture experiments were used for mechanistic studies, and quantitative polymerase chain reaction, microarrays, ELISA, and immunohistochemistry were used to analyze the cardiac phenotypes. RESULTS: Both EC deletion of VEGF receptor (VEGFR)-1 and adeno-associated viral vector-mediated delivery of the VEGFR1-specific ligands VEGF-B or placental growth factor into the myocardium increased the coronary vasculature and induced CMC hypertrophy in adult mice. The resulting cardiac hypertrophy was physiological, as indicated by preserved cardiac function and exercise capacity and lack of pathological gene activation. These changes were mediated by increased VEGF signaling via endothelial VEGFR2, because the effects of VEGF-B and placental growth factor on both angiogenesis and CMC growth were fully inhibited by treatment with antibodies blocking VEGFR2 or by endothelial deletion of VEGFR2. To identify activated pathways downstream of VEGFR2, whole-genome transcriptomics and secretome analyses were performed, and the Notch and ErbB pathways were shown to be involved in transducing signals for EC-CMC cross talk in response to angiogenesis. Pharmacological or genetic blocking of ErbB signaling also inhibited part of the VEGF-B-induced effects in the heart. CONCLUSIONS: This study reveals that cross talk between the EC VEGFR2 and CMC ErbB signaling pathways coordinates CMC hypertrophy with angiogenesis, contributing to physiological cardiac growth.

Heparin-binding epidermal growth factor (EGF)-like growth factor in pediatric patients with type 1 diabetes mellitus.

Heparin-binding EGF-like growth factor (HB-EGF) is involved in atherosclerosis progression. We investigated association between plasma HB-EGF levels and lipid, oxidative stress and inflammatory biomarkers in pediatric patients with type 1 diabetes mellitus (T1DM). Levels of HB-EGF, high-sensitive C-reactive protein (hsCRP), prooxidant-antioxidant balance (PAB), total antioxidant status (TAS), oxidized low-density lipoproteins (oxLDL), metabolic control and serum lipid parameters and paraoxonase 1 (PON1) activity were determined in 74 patients and 40 controls. In comparison to controls, patients had significantly higher levels (p < 0.01) of HB-EGF, hsCRP, PAB and oxLDL particles (p < 0.001), but lower levels of TAS and PON1 activity. In T1DM group, HB-EFG levels were positively associated with hsCRP, PAB and oxLDL levels. hsCRP and oxLDL levels were independent predictors of HB-EGF concentration. We demonstrated that oxidative modifications of LDL particles and low-grade inflammation are main determinants of increased plasma HB-EGF levels, which indicates an interactive role of oxidative stress, dyslipidemia and inflammation.

Attenuating influenza a virus infection by heparin binding EGF-like growth factor.

Cell entry of influenza A virus (IAV) was reported to be promoted by epidermal growth factor receptor (EGFR). On the other hand, binding of heparin-binding EGF-like growth factor (HB-EGF) to EGFR leads to internalisation and degradation of the receptors. This study aimed to testify whether or not HB-EGF-induced downregulation of EGFR could attenuate IAV cell entry and subsequently diminish the infection. Immunoblotting and plaque assay revealed that HB-EGF-induced degradation of EGFR led to reduction of viral matrix 1 protein level and suppressed virion production. In addition, immunoblotting and imaging flow cytometric analysis demonstrated that IAV-induced phosphorylation of STAT1 and its localisation to nucleus in the early stage of infection were inhibited by HB-EGF treatment. This suggested the potential of HB-EGF in modulating uncontrolled and exaggerated inflammatory response caused by IAV infection. Together these findings attest the potential of HB-EGF mediated endocytosis and degradation of EGFR as a novel anti-IAV strategy.

Phase I study of the anti-heparin-binding epidermal growth factor-like growth factor antibody U3-1565 with cetuximab in patients with cetuximab- or panitumumab-resistant metastatic colorectal cancer.

KRAS wild-type colorectal cancers initially responsive to anti-endothelial growth factor receptor (EGFR) antibodies [cetuximab (Cetu)/panitumumab (Pani)] develop acquired resistance. Overexpression of EGFR ligands such as heparin-binding EGF-like growth factor (HB-EGF) may be one resistance mechanism. This phase I study of U3-1565, anti-HB-EGF antibody, and Cetu combination therapy enrolled patients with KRAS wild-type metastatic colorectal cancer who had received two ≤ regimens with fluoropyrimidine, oxaliplatin, irinotecan, and Cetu/Pani and had disease progression on Cetu/Pani. Recommended dose (RD) was determined in the 1st stage, followed by evaluation of efficacy at the RD level in the 2nd-stage. Cetu was given at a loading dose of 400 mg/m2 followed by weekly infusions of 250 mg/m2 in levels 1 and 0. U3-1565 was administered at a loading dose of 24 mg/m2 followed by biweekly infusions of 16 mg/m2 in level 1 and 16-12 mg/m2 in level 0. Twenty-two patients were enrolled. No dose-limiting toxicities were observed among three patients in level 1 in the first stage, which was determined as RD. Grade 3 or higher adverse events occurred in 59.1%; those in ≥5% of patients were anemia, γ-GTP elevation, and acneiform rash. Overall response rate was 0.0% [95% confidence interval (CI): 0.0%-15.4%] and disease control was achieved in 17 patients (77.3%, 95% CI: 54.6%-92.2%). Median progression-free survival time was 85.0 days (95% CI: 54.0-91.0) and median survival time was 196 days (95% CI: 113.0-306.0). RD was determined as level 1. The efficacy of this combination therapy after progression on Cetu/Pani was negligible. Trial Registration: UMIN000013006.

IL-13 signaling through IL-13 receptor α2 mediates airway epithelial wound repair.

Asthma is an airway inflammatory disease characterized by epithelial barrier dysfunction and airway remodeling. Interleukin-13 (IL-13) is a pleiotropic cytokine shown to contribute to features of airway remodeling. We have previously demonstrated that IL-13 is an important mediator of normal airway epithelial repair and health. The role of IL-13 signaling via its receptor subunits (IL-13Rα1/IL-4Rα and IL-13Rα2) in airway epithelial repair and restoration of intact barrier function is not well understood and was investigated in this study using in vitro models. The blocking of IL-13 signaling via IL-13Rα2 significantly reduced airway epithelial repair by 24 h post-mechanical wounding in 1HAEo- cells. Expression and release of repair-mediating growth factor, heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF), and subsequent activation of EGF receptor (EGFR) were also significantly reduced in response to wounding when IL-13Rα2 was blocked. Our data support that IL-13 signals via IL-13Rα2 to mediate normal airway epithelial repair via HB-EGF-dependent activation of EGFR. In human donor lung tissues, we observed that airway epithelium of asthmatics expressed significantly decreased levels of IL-13Rα2 and increased levels of IL-13Rα1 compared with nonasthmatics. Dysregulated expression of IL-13 receptor subunits in the airways of asthmatics may thus contribute to the epithelial barrier dysfunction observed in asthma.-Yang, S. J., Allahverdian, S., Saunders, A. D. R., Liu, E., Dorscheid, D. R. IL-13 signaling through IL-13 receptor α2 mediates airway epithelial wound repair.

Super-resolution microscopy unveils transmembrane domain-mediated internalization of cross-reacting material 197 into diphtheria toxin-resistant mouse J774A.1 cells and primary rat fibroblasts in vitro.

Diphtheria toxin (DT) efficiently inhibits protein synthesis in human cells, resulting in severe disease diphtheria. The sensitivity towards DT varies between mammalian species. Mice and rats are resistant to DT. However, the reason underlying this insensitivity is controversially discussed and not well understood. Therefore, we investigated the steps of DT uptake, i.e. receptor binding and internalization into mouse J774A.1 macrophages and primary rat fibroblasts. We exploited the non-toxic DT-mutant cross-reacting material 197 (CRM197) and three additional receptor binding-deficient mutants (250 nM each) to investigate binding to cell surface and internalization into murine cells via flow cytometry and stimulated emission depletion (STED) super-resolution optical microscopy. Dual-color STED imaging unveiled CRM197 interacting with the murine precursor of the heparin-binding epidermal growth factor-like growth factor (HB-EGF). Moreover, we identified CRM197's transmembrane domain as an additional HB-EGF binding site, which is also involved in the receptor-mediated internalization into murine cells. However, we do not find evidence for translocation of the catalytically active subunit (DTA) into the cytosol when 250 nM DT were applied. In conclusion, we provide evidence that the resistance of murine cells to DT is caused by an insufficiency of DTA to escape from endosomes and reach the cytosol. Possibly, a higher affinity interaction of DT and the HB-EGF is required for translocation, which highlights the role of the receptor in the endosomes during the translocation step. We extend the current knowledge about cellular uptake of the medically relevant DT and CRM197.

Activation of Pyk2 by CaM kinase II in cultured hypothalamic neurons and gonadotroph cells.

Gonadotropin-releasing hormone (GnRH) is secreted from hypothalamic GnRH neurons and stimulates a GnRH receptor in gonadotroph cells and GnRH neurons. The GnRH receptor belongs to the G-protein-coupled receptors, and stimulation of the GnRH receptor activates extracellular signal-regulated protein kinase (ERK). We reported previously that the δ2 isoform of Ca2+ /calmodulin-dependent protein kinase II (CaM kinase IIδ2) was involved in GnRH-induced ERK activation in cultured GnRH neurons (GT1-7 cells). Recently, we found that GnRH treatment of GT1-7 cells activated proline-rich tyrosine kinase 2 (Pyk2), and Pyk2 was involved in ERK activation. In the current study, we examined the possibility that CaM kinase IIδ2 might activate Pyk2. Knockdown of CaM kinase IIδ2 and KN93, an inhibitor of CaM kinases, inhibited the GnRH-induced activation of Pyk2. In the case of cultured gonadotroph cells (αT3-1 cells), knockdown of CaM kinase IIß'e inhibited GnRH-induced Pyk2 activation. In addition, our inhibitor studies indicated that Pyk2 and CaM kinase II were involved in the GnRH-induced shedding of proHB-EGF in GT1-7 cells. These results suggested that CaM kinase II activated the ERK pathway through Pyk2 activation and HB-EGF production in response to GnRH.