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Cells respond to mechanical stimuli with altered signaling networks. Here, we show that mechanical forces rapidly induce phosphorylation of CD97/ADGRE5 (pCD97) at its intracellular C-terminal PDZ-binding motif (PBM). Biochemically, this phosphorylation disrupts CD97 binding to PDZ domains of the scaffold protein DLG1. In shear-stressed cells, pCD97 appears not only in junctions, retracting fibers, and the attachment area but also in lost membrane patches, demonstrating (intra)cellular detachment at the CD97 PBM. This motif is critical for the CD97-dependent mechanoresponse. Cells expressing CD97 without the PBM are more deformable, and under shear stress, these cells lose cell contacts faster and show changes in the actin cytoskeleton when compared with cells expressing full-length CD97. Our data indicate CD97 linkage to the cytoskeleton. Consistently, CD97 knockout phenocopies CD97 without the PBM, and membranous CD97 is organized in an F-actin-dependent manner. In summary, CD97 shapes the cellular mechanoresponse through signaling modulation via its PBM. • Phosphorylation of S740 in the PDZ-binding motif (PBM) of CD97 alters its binding • Mechanical force-induced phospho-CD97 at S740 facilitates (intra)cellular detachment • Protein kinases C and D can phosphorylate CD97 at S740 • PBM- and GNA12/13-mediated signaling of CD97 are partly independent Adhesion GPCRs are potential mechanoreceptors. Hilbig et al. demonstrate that CD97/ADGRE5 shapes the cellular mechanoresponse by modulating signaling at its intracellular tail. Mechanical stimuli rapidly induce phosphorylation in the CD97 C-terminal PDZ-binding motif, which alters the intracellular coupling of the receptor, thereby affecting cellular viscoelastic properties and detachment. [ABSTRACT FROM AUTHOR]

Objective Variants in Proprotein Convertase Subtilisin/Kexin Type 1 ( PCSK1 ) may be causative for obesity as suggested by monogenic cases and association studies. Here we assessed the functional relevance in experimental studies and the clinical relevance through detailed metabolic phenotyping of newly identified and known PCSK1 variants in children. Results In 52 obese children selected for elevated proinsulin levels and/or impaired glucose tolerance, we found eight known variants and two novel heterozygous variants (c.1095 + 1G > A and p.S24C) by sequencing the PCSK1 gene. Patients with the new variants presented with extreme obesity, impaired glucose tolerance, and PCOS. Functionally, c.1095 + 1G > A caused skipping of exon8 translation and a complete loss of enzymatic activity. The protein was retained within the endoplasmic reticulum (ER) causing ER stress. The p.S24C variant had no functional effect on protein size, cell trafficking, or enzymatic activity. The known variants rs6230, rs35753085, and rs725522 in the 5′ end did not affect PCSK1 promoter activity. In clinical association studies in 1673 lean and obese children, we confirmed associations of rs6232 and rs6234 with BMI-SDS and of rs725522 with glucose stimulated insulin secretion and Matsuda index. We did not find the new variants in any other subjects. Conclusions We identified and functionally characterized two rare novel PCSK1 variants of which c.1095 + 1G > A caused complete loss of protein function. In addition to confirming rs6232 and rs6234 in PCSK1 as polygenic risk variants for childhood obesity, we describe an association of rs725522 with insulin metabolism. Our results support the contribution of PCSK1 variants to obesity predisposition in children. [ABSTRACT FROM AUTHOR]

Abstract: Background and aims: To understand biomechanics of ligaments and tendons data on their material properties are necessary. The iliotibial tract is a suitable model for virtual pelvic or lower extremity ligaments due to its parallel fibers, which facilitates biomechanical testing. Here, we determined Young''s modulus (YM) as secant stiffness between defined limits of the iliotibial tract and correlated the data to ultimate stress (US) of the specimens and to age, gender and body weight of the body donors. Materials and methods: Thirty eight specimens from 12 iliotibial tracts of 10 young donors (mean age 31.2±9.1 years) were investigated biomechanically. After preconditioning, YM were determined in the ranges of 0–4 and 4–11N/mm² of applied stress and from 4N/mm² of applied stress to US. Results: YM of the specimens were 84.7±30.2 (0–4N/mm²), 335.4±101.9 (4–11N/mm²), and 369.1±191.5 (4N/mm² to US) N/mm², respectively. The mean US was 35.8±16.4N/mm². YM and US correlated closely in the ranges of 4–11N/mm² (r=0.95) and 4N/mm² to US (r=0.91). YM did not correlate to age, body weight or gender within these young donors. Concerning tissue behavior a decrease of YM, i.e. weakening, is more common than an increase of YM, i.e. stiffening, before specimen failure. Overall, YM of specimens from young donors were significantly lower compared to those of old donors. Discussion and conclusions: This is the first study providing age-dependent nonlinear stiffness properties of the iliotibial tract. YM is significantly lower in young than in old donors and is thus a subject of alteration during life time. [Copyright &y& Elsevier]

Abstract: Obesity is associated with accelerated atherosclerosis. Adipokines may directly influence vessel wall homeostasis by influencing the function of endothelial cells, arterial smooth muscle cells, and modulating inflammation. Recently, visceral adipose tissue-derived serpin (vaspin) was identified as a novel adipokine related to obesity and its metabolic consequences. However, the regulation of vaspin serum concentrations in human atherosclerosis is unknown. We therefore assessed vaspin serum concentrations in 107 consecutive patients with carotid stenosis undergoing carotid endarterectomy (CEA) in relation to severity of atherosclerosis, measures of obesity and circulating markers of obesity and atherosclerosis. Vaspin serum concentrations were significantly lower in patients with carotid stenosis who experienced an ischemic event within 3 months before surgery compared to asymptomatic patients. However, circulating vaspin was not associated with measures of atherosclerosis severity as maximum percentage stenosis. Vaspin serum concentrations were indistinguishable before and after CEA. We found a significant correlation between vaspin and leptin serum concentrations supporting previous results that vaspin closely reflects body fat mass. In conclusion, our data show that low vaspin serum concentrations correlate with recently experienced ischemic events in patients with carotid stenosis despite the lack of an association between circulating vaspin and parameters of atherosclerosis severity. [Copyright &y& Elsevier]

Summary: The inhibition of vascular smooth muscle cell proliferation is mediated through two intracellular estrogen receptors (ERs), ER-? and ER-?. Deletion variants of ER-? have been decribed for cultures of smooth muscle cells. The internal thoracic artery is frequently used as coronary artery bypass graft, yet neither has it been studied for the expression of ER subtypes nor for the synthesis of the ERs in morphologically hetergeneous smooth muscle cells. Using nested RT-PCR, we have demonstrated the mRNA for ER-? splicing variants in intact human internal thoracic arteries. The 7? deletion variant occurred in 8 out of 12 cases, the full-length transcript in three cases. The full-length transcript was always found for the ER-?. Immunolocalization revealed ER-positive nuclei in the desmin-positive subset of smooth muscle cells, but not in cytokeratin (CK)-positive cells of the thickened intima. Morphological evidence is presented suggesting that ER synthesis is high in the tunica media when cell proliferation of smooth muscle cells is increased. We conclude that, in internal thoracic arteries, the 7? deletion variant of ER-? occurs in 75%, whereas the full-length transcript is found in all cases. The significance remains unclear. [Copyright &y& Elsevier]

The Adhesion G protein-coupled receptor (GPCR) CD97/ADGRE5 is induced, upregulated, and/or biochemically modified in various malignancies, compared to the corresponding normal tissues. As tumor cells are generally more resistant to apoptosis, we here studied the ability of CD97 to regulate tumor cell survival under apoptotic conditions. Stable overexpression of wild-type CD97 reduced serum starvation- and staurosporine-induced intrinsic and tumor necrosis factor (TNF)/cycloheximide-induced extrinsic apoptosis, indicated by an increase in cell viability, a lower percentage of cells within the subG0/G1 phase, expressing annexin V, or having condensed nuclei, and a reduction of DNA laddering. Protection from cell death by CD97 was accompanied by an inhibition of caspase activation and modulation of anti- and pro-apoptotic members of the BCL-2 superfamily. shRNA-mediated knockdown of CD97 and, in part, truncation of the seven-span transmembrane (TM7) region of CD97 increased caspase-mediated apoptosis. Protection from apoptosis required not only the TM7 region but also cleavage of the receptor at its GPCR proteolysis site (GPS), whereas alternative splicing of its extracellular domain had no effect. Together, our data indicate a role of CD97 in tumor cell survival. [ABSTRACT FROM AUTHOR]

Abstract: The EGF-TM7 receptor CD97 shows different features of expression and function in muscle cells compared to hematopoetic and tumor cells. Since the molecular function and regulation of CD97 are poorly understood, this study aimed at defining its basal transcriptional regulation in smooth muscle cells (SMCs). The computational analysis of the CD97 5′-flanking region revealed that the TATA box-lacking promoter possesses several GC-rich regions as putative Sp1/Sp3 binding sites. Transfection studies with serially deleted promoter constructs demonstrated that the minimal promoter fragment resided in the −218/+45 region containing one out of five identified GC-boxes in the leiomyosarcoma cell line SK-LMS-1 and human bronchial smooth muscle cells (HbSMCs). Mutation of the most proximal GC-site in CD97 reporter gene constructs caused a significant decrease in promoter activity. Gel shift assays and chromatin immunoprecipitation revealed that Sp1 and Sp3 bound specifically to the most proximal GC-site. Furthermore, we showed that Sp1 and Sp3 over-expression activates CD97 promoter activity in HEK293 cells. Our data characterize for the first time the activity of the human CD97 promoter which is controlled by Sp1/Sp3 transcription factors in SMCs. [Copyright &y& Elsevier]

CD97, a cell surface molecule on immune cells with potential adhesive function, is a member of the epidermal growth factor seven-span transmembrane (EGF-TM7) family. We have cloned and characterized bovine CD97 and determined its expression in various cells and tissues. Based on sequence alignment with human genomic DNA, as well as human cDNA sequences encoding various CD97 isoforms, we predict that bovine CD97 mRNA occurs in four splice variants. The encoded CD97 isoforms (800, 751, 756, and 707 amino acids in length) contain different numbers of EGF-like modules, a stalk region, and a TM7 domain with a cytoplasmic tail. RT-PCR demonstrated expression of CD97 mRNA in leukocytes and several other tissues. In each cell type, CD97 mRNA encoding the shortest isoform, CD97 (EGF 1,2,5), was detected at the highest level, which was consistent with the cell surface expression of a 90-kDa polypeptide precipitated from lysates of peripheral blood mononuclear cells. [Copyright &y& Elsevier]