Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

The vitally important skin barrier is formed by extensive cross-linking activity of transglutaminases (TGs) during terminal epidermal differentiation. We have previously shown that epidermal deficiency of a disintegrin and metalloproteinase 17 (ADAM17), the principal EGFR ligand sheddase, results in postnatal skin barrier defects in mice due to impeded TG activity. However, the mechanism by which ADAM17/EGFR signalling maintains TG activity during epidermal differentiation remains elusive. Here we demonstrate that ADAM17-dependent EGFR signalling promotes TG activity in keratinocytes committed to terminal differentiation by direct induction of TG1 expression. Restored TG1 expression of EGF-stimulated differentiated Adam17 −/− keratinocytes was strongly repressed by inhibitors for PLCγ1 or protein kinase C (PKC) pathways, while treatment with the PKC stimulator 12- O -tetradecanoylphorbol-13-acetate restored TG activity in the epidermis of keratinocyte-specific Adam17 −/− ( AD17 Δ KC ) mice. Further investigations emphasized the expression of PKCη, a mediator of TGM1 transcription, to be sensitive to EGFR activation. In agreement, topical skin application of cholesterol sulfate, an activator of PKCη, significantly improved TG activity in epidermis of AD17 Δ KC mice. Our results suggest ADAM17/EGFR-driven PLCγ1 and PKC pathways as important promoters of TG1 expression during terminal keratinocyte differentiation. These findings may help to identify new therapeutic targets for inflammatory skin diseases related to epidermal barrier defects.

Keratinocyte-specific deletion of ADAM17 in mice impairs terminal differentiation of keratinocytes leading to severe epidermal barrier defects. Mice deficient for ADAM17 in keratinocytes phenocopy mice with a keratinocyte-specific deletion of epidermal growth factor receptor (EGFR), which highlights the role of ADAM17 as a "ligand sheddase" of EGFR ligands. In this study, we aim for the first proteomic/degradomic approach to characterize the disruption of the ADAM17-EGFR signaling axis and its consequences for epidermal barrier formation. Proteomic profiling of the epidermal proteome of mice deficient for either ADAM17 or EGFR in keratinocytes at postnatal days 3 and 10 revealed highly similar protein alterations for ADAM17 and EGFR deficiency. These include massive proteome alterations of structural and regulatory components important for barrier formation such as transglutaminases, involucrin, filaggrin, and filaggrin-2. Cleavage site analysis using terminal amine isotopic labeling of substrates revealed increased proteolytic processing of S100 fused-type proteins including filaggrin-2. Alterations in proteolytic processing are supported by altered abundance of numerous proteases upon keratinocyte-specific Adam17 or Egfr deletion, among them kallikreins, cathepsins, and their inhibitors. This study highlights the essential role of proteolytic processing for maintenance of a functional epidermal barrier. Furthermore, it suggests that most defects in formation of the postnatal epidermal barrier upon keratinocyte-specific ADAM17 deletion are mediated via EGFR.

Atherosclerosis is a chronic inflammation of the arterial wall caused by accumulation of cholesterol. This complex process involves endothelial and smooth muscle cells as well as the recruitment and differentiation of circulating monocytes. Integrins are membrane-bound molecules that are involved in signaling between cells and the extracellular matrix, thereby influencing intracellular signaling and cytoskeletal stability. We and others have described the in vivo consequences of germline deletion of the β3 integrin in inflammation, atherosclerosis and cancer. However, the distinct pathophysiological consequences of its tissue-specific deletion in macrophage function in development of atherosclerotic lesions remain mostly elusive. Our aim was to characterize for the first time the phenotype of mice with tissue-specific deletion of β3 integrin in macrophages (LyzMCre) under high fat diet (HFD) condition on atherosclerotic prone backgrounds (ApoE-/- and LDLr-/-), and to elucidate the signaling mechanism involved using in vitro methods. Tissue specific β3 integrin deficiency in macrophages alone is sufficient to cause increased lesion formation in the aorta of mice on HFD on both backgrounds used suggesting an important atheroprotective role played by this integrin. β3 integrin-deficient bone marrow-derived macrophages (BMDM) treated with oxLDL in vitro demonstrated a strong cholesterol uptake and increased foam cell formation. We identified that β3 integrin deficiency in macrophages caused an early activation of Ras followed by ERK phosphorylation, deficient cholesterol efflux and decreased expression of cholesterol transporter (ABCA1) which is a major regulator of cellular cholesterol. Inhibition of the ERK pathway reduced foam cell formation of macrophages at least partially by restoring expression of ABCA1. Taken together, our results show that macrophage β3 integrin is an important signaling molecule for cellular activation. Its deficiency showed important functional consequences in regard to inflammation and atherosclerosis that could be possibly modulated by interfering with downstream signaling.

CONTEXT:Primary macronodular adrenal hyperplasia (PMAH) is a rare cause of Cushing's syndrome, which may present in the context of different familial multitumor syndromes. Heterozygous inactivating germline mutations of armadillo repeat containing 5 (ARMC5) have very recently been described as cause for sporadic PMAH. Whether this genetic condition also causes familial PMAH in association with other neoplasias is unclear.OBJECTIVE:The aim of the present study was to delineate the molecular cause in a large family with PMAH and other neoplasias.PATIENTS AND METHODS:Whole-genome sequencing and comprehensive clinical and biochemical phenotyping was performed in members of a PMAH affected family. Nodules derived from adrenal surgery and pancreatic and meningeal tumor tissue were analyzed for accompanying somatic mutations in the identified target genes.RESULTS:PMAH presenting either as overt or subclinical Cushing's syndrome was accompanied by a heterozygous germline mutation in ARMC5 (p.A110fs*9) located on chromosome 16. Analysis of tumor tissue showed different somatic ARMC5 mutations in adrenal nodules supporting a second hit hypothesis with inactivation of a tumor suppressor gene. A damaging somatic ARMC5 mutation was also found in a concomitant meningioma (p.R502fs) but not in a pancreatic tumor, suggesting biallelic inactivation of ARMC5 as causal also for the intracranial meningioma.CONCLUSIONS:Our analysis further confirms inherited inactivating ARMC5 mutations as a cause of familial PMAH and suggests an additional role for the development of concomitant intracranial meningiomas.