The carboxyl-terminal TSP1-homology domain is the biologically active effector peptide of matricellular protein CCN5 that counteracts profibrotic CCN2.

Cellular Communication Network (CCN) proteins have multimodular structures important for their roles in cellular responses associated with organ development and tissue homeostasis. CCN2 has previously been reported to be secreted as a preproprotein that requires proteolytic activation to release its bioactive carboxyl-terminal fragment. Here, our goal was to resolve whether CCN5, a divergent member of the CCN family with converse functions relative to CCN2, releases the TSP1 homology domain as its bioactive signaling entity. The recombinant CCN5 or CCN3 TSP1 homology domains were produced in ExpiCHO-S or DG44 CHO cells as secretory fusion proteins appended to the carboxyl-terminal end of His-Halo-Sumo or amino-terminal end of human albumin and purified from the cell culture medium. We tested these fusion proteins in various phosphokinase signaling pathways or cell physiologic assays. Fusion proteins with the CCN5 TSP1 domain inhibited key signaling pathways previously reported to be stimulated by CCN2, irrespective of fusion partner. The fusion proteins also efficiently inhibited CCN1/2-stimulated cell migration and gap closure following scratch wound of fibroblasts. Fusion protein with the CCN3 TSP1 domain inhibited these functions with similar efficacy and potency as that of the CCN5 TSP1 domain. The CCN5 TSP1 domain also recapitulated a positive regulatory function previously assigned to full-length CCN5, that is, induction of estrogen receptor-α mRNA expression in triple negative MDA-MB-231 mammary adenocarcinoma cells and inhibited epithelial-to-mesenchymal transition and CCN2-induced mammosphere formation of MCF-7 adenocarcinoma cells. In conclusion, the CCN5 TSP1 domain is the bioactive entity that confers the biologic functions of unprocessed CCN5.

Connective tissue growth factor (CCN2) is a matricellular preproprotein controlled by proteolytic activation.

Connective tissue growth factor (CTGF; now often referred to as CCN2) is a secreted protein predominantly expressed during development, in various pathological conditions that involve enhanced fibrogenesis and tissue fibrosis, and in several cancers and is currently an emerging target in several early-phase clinical trials. Tissues containing high CCN2 activities often display smaller degradation products of full-length CCN2 (FL-CCN2). Interpretation of these observations is complicated by the fact that a uniform protein structure that defines biologically active CCN2 has not yet been resolved. Here, using DG44 CHO cells engineered to produce and secrete FL-CCN2 and cell signaling and cell physiological activity assays, we demonstrate that FL-CCN2 is itself an inactive precursor and that a proteolytic fragment comprising domains III (thrombospondin type 1 repeat) and IV (cystine knot) appears to convey all biologically relevant activities of CCN2. In congruence with these findings, purified FL-CCN2 could be cleaved and activated following incubation with matrix metalloproteinase activities. Furthermore, the C-terminal fragment of CCN2 (domains III and IV) also formed homodimers that were ∼20-fold more potent than the monomeric form in activating intracellular phosphokinase cascades. The homodimer elicited activation of fibroblast migration, stimulated assembly of focal adhesion complexes, enhanced RANKL-induced osteoclast differentiation of RAW264.7 cells, and promoted mammosphere formation of MCF-7 mammary cancer cells. In conclusion, CCN2 is synthesized and secreted as a preproprotein that is autoinhibited by its two N-terminal domains and requires proteolytic processing and homodimerization to become fully biologically active.

Lactate receptor sites link neurotransmission, neurovascular coupling, and brain energy metabolism.

The G-protein-coupled lactate receptor, GPR81 (HCA1), is known to promote lipid storage in adipocytes by downregulating cAMP levels. Here, we show that GPR81 is also present in the mammalian brain, including regions of the cerebral neocortex and hippocampus, where it can be activated by physiological concentrations of lactate and by the specific GPR81 agonist 3,5-dihydroxybenzoate to reduce cAMP. Cerebral GPR81 is concentrated on the synaptic membranes of excitatory synapses, with a postsynaptic predominance. GPR81 is also enriched at the blood-brain-barrier: the GPR81 densities at endothelial cell membranes are about twice the GPR81 density at membranes of perivascular astrocytic processes, but about one-seventh of that on synaptic membranes. There is only a slight signal in perisynaptic processes of astrocytes. In synaptic spines, as well as in adipocytes, GPR81 immunoreactivity is located on subplasmalemmal vesicular organelles, suggesting trafficking of the protein to and from the plasma membrane. The results indicate roles of lactate in brain signaling, including a neuronal glucose and glycogen saving response to the supply of lactate. We propose that lactate, through activation of GPR81 receptors, can act as a volume transmitter that links neuronal activity, cerebral energy metabolism and energy substrate availability.

Connective tissue growth factor/CCN2 attenuates ß-adrenergic receptor responsiveness and cardiotoxicity by induction of G protein-coupled receptor kinase-5 in cardiomyocytes.

Myocardial connective tissue growth factor (CTGF/CCN2) is induced in heart failure, a condition associated with diminution of ß-adrenergic receptor (ß-AR) responsiveness. Accordingly, we aimed to investigate whether CTGF could play a mechanistic role in regulation of ß-AR responsiveness. Concentration-response curves of isoproterenol-stimulated cAMP generation in cardiomyocytes from transgenic mice with cardiac-restricted overexpression of CTGF (Tg-CTGF) or cardiomyocytes pretreated with recombinant human CTGF (rec-hCTGF) revealed marked reduction of both ß1-AR and ß2-AR responsiveness. Consistently, ventricular muscle strips from Tg-CTGF mice stimulated with isoproterenol displayed attenuation of maximal inotropic responses. However, no differences of maximal inotropic responses of myocardial fibers from Tg-CTGF mice and nontransgenic littermate control (NLC) mice were discerned when stimulated with supramaximal concentrations of dibutyryl-cAMP, indicating preserved downstream responsiveness to cAMP. Congruent with a mechanism of desensitization of ß-ARs, mRNA and protein levels of G protein-coupled receptor kinase 5 (GRK5) were found isoform-selective upregulated in both cardiomyocytes from Tg-CTGF mice and cardiomyocytes exposed to rec-hCTGF. Corroborating a mechanism of GRK5 in CTGF-mediated control of ß-AR sensitivity, Chinese hamster ovary cells pretreated with rec-hCTGF displayed increased agonist- and biased ligand-stimulated ß-arrestin binding to ß-ARs. Despite increased sensitivity of cardiomyocytes from GRK5-knockout (KO) mice to ß-adrenergic agonists, pretreatment of GRK5-KO cardiomyocytes with rec-hCTGF, as opposed to cardiomyocytes from wild-type mice, did not alter ß-AR responsiveness. Finally, Tg-CTGF mice subjected to chronic exposure (14 days) to isoproterenol revealed blunted myocardial hypertrophy and preserved cardiac function versus NLC mice. In conclusion, this study uncovers a novel mechanism controlling ß-AR responsiveness in cardiomyocytes involving CTGF-mediated regulation of GRK5.

Tissue distribution and transcriptional regulation of CCN5 in the heart after myocardial infarction.

CCN5 is a divergent member of the cellular communication network factor (CCN) family in that it lacks the carboxyl terminal cystine knot domain common to the other CCN family members. CCN5 has been reported to antagonize the profibrotic actions of CCN2 and to inhibit myocardial collagen deposition and fibrosis in chronic pressure overload of the heart. However, what mechanisms that regulate CCN5 activity in the heart remain unknown. Recombinant, replication defective adenovirus encoding firefly luciferase under control of the human CCN5 promoter was prepared and used to investigate what mechanisms regulate CCN5 transcription in relevant cells. Tissue distribution of CCN5 in hearts from healthy mice and from mice subjected to myocardial infarction was investigated. Contrary to the profibrotic immediate early gene CCN2, we find that CCN5 is induced in the late proliferation and maturation phases of scar healing. CCN5 was identified principally in endothelial cells, fibroblasts, smooth muscle cells, and macrophages. Our data show that CCN5 gene transcription and protein levels are induced by catecholamines via ß2-adrenergic receptors. Myocardial induction of CCN5 was further confirmed in isoproterenol-infused mice. We also find that CCN5 transcription is repressed by TNF-α, an inflammatory mediator highly elevated in early phases of wound healing following myocardial infarction. In conclusion, CCN5 predominates in endothelial cells, fibroblasts, and macrophages of the differentiating scar tissue and its transcription is conversely regulated by ß2-adrenergic agonists and TNF-α.

Adrenomedullin is increased in alveolar macrophages and released from the lungs into the circulation in severe heart failure.

Adrenomedullin (AM) is a potent vasorelaxing peptide with natriuretic, diuretic, and growth inhibitory properties. Plasma concentrations and myocardial AM expression are increased in heart failure (HF). Since AM and AM binding sites are abundantly expressed in the lungs, we investigated to what extent pulmonary AM and AM receptor subtypes [CRLR/RAMP2 (AM1) and CRLR/RAMP3 (AM2)] are changed in HF and whether the lungs contribute to the increased plasma concentrations of AM reported in HF. Pulmonary AM mRNA and protein expression were increased by 2.8- and 2.6-fold, respectively, whereas mRNA expression of RAMP2 and CRLR was decreased in rats with HF 7 days after induction of MI compared to sham-operated rats (P < 0.05). Pulmonary AM receptor density was substantially decreased in HF rats compared to sham (3.7 +/-0.6 vs. 29.9 +/- 1.1 fmol/mg membrane protein; P < 0.05). Immunoreactivities against AM and the AM receptor components CRLR, RAMP2, and RAMP3 in the pulmonary tissue were seen in vascular smooth muscle cells, vascular endothelial cells, and in alveolar macrophages. AM mRNA expression in alveolar macrophages obtained from HF rats by bronchoalveolar lavage was 2.9-fold higher than in sham-operated rats (P < 0.05). An even more substantial increase of AM mRNA expression was found in alveolar macrophages from patients with HF (10-fold, P < 0.05), and this increase displayed a negative correlation to left ventricular systolic function (P < 0.05). Furthermore, a net release of AM from the lungs into the circulation was only found in HF patients with the most severe left ventricular systolic dysfunction. Thus, our data demonstrate increased expression and decreased receptor binding of AM in the lungs in severe HF. Furthermore, our data indicate that alveolar macrophages are an important source of pulmonary AM in both experimental and clinical HF. Finally, a net release of AM from the lungs into the circulation was only found in patients with severe systolic dysfunction.

Preclinical Combination Studies of an FGFR2 Targeted Thorium-227 Conjugate and the ATR Inhibitor BAY 1895344.

PURPOSE: Fibroblast growth factor receptor 2 (FGFR2) has been previously reported to be overexpressed in several types of cancer, whereas the expression in normal tissue is considered to be moderate to low. Thus, FGFR2 is regarded as an attractive tumor antigen for targeted alpha therapy. This study reports the evaluation of an FGFR2-targeted thorium-227 conjugate (FGFR2-TTC, BAY 2304058) comprising an anti-FGFR2 antibody, a chelator moiety covalently conjugated to the antibody, and the alpha particle-emitting radionuclide thorium-227. FGFR2-TTC was assessed as a monotherapy and in combination with the DNA damage response inhibitor ATRi BAY 1895344. METHODS AND MATERIALS: The in vitro cytotoxicity and mechanism of action were evaluated by determining cell viability, the DNA damage response marker γH2A.X, and cell cycle analyses. The in vivo efficacy was determined using human tumor xenograft models in nude mice. RESULTS: In vitro mechanistic assays demonstrated upregulation of γH2A.X and induction of cell cycle arrest in several FGFR2-expressing cancer cell lines after treatment with FGFR2-TTC. In vivo, FGFR2-TTC significantly inhibited tumor growth at a dose of 500 kBq/kg in the xenograft models NCI-H716, SNU-16, and MFM-223. By combining FGFR2-TTC with the ATR inhibitor BAY 1895344, an increased potency was observed in vitro, as were elevated levels of γH2A.X and inhibition of FGFR2-TTC-mediated cell cycle arrest. In the MFM-223 tumor xenograft model, combination of the ATRi BAY 1895344 with FGFR2-TTC resulted in significant tumor growth inhibition at doses at which the single agents had no effect. CONCLUSIONS: The data provide a mechanism-based rationale for combining the FGFR2-TTC with the ATRi BAY 1895344 as a new therapeutic approach for treatment of FGFR2-positive tumors from different cancer indications.

CTGF/CCN2 Postconditioning Increases Tolerance of Murine Hearts towards Ischemia-Reperfusion Injury.

BACKGROUND AND PURPOSE: Previous studies of ischemia-reperfusion injury (IRI) in hearts from mice with cardiac-restricted overexpression of CCN2 have shown that CCN2 increases tolerance towards IRI. The objectives of this study were to investigate to what extent post-ischemic administration of recombinant human CCN2 (rhCCN2) would limit infarct size and improve functional recovery and what signaling pathways are involved. EXPERIMENTAL APPROACH: Isolated mice hearts were perfused ad modum Langendorff, subjected to no-flow, global ischemia, and subsequently, exposed to mammalian cell derived, full-length (38-40kDa) rhCCN2 (250 nM) or vehicle during the first 15 min of a 60 min reperfusion period. KEY RESULTS: Post-ischemic administration of rhCCN2 resulted in attenuation of infarct size from 58 ± 4% to 34 ± 2% (p < 0.001) which was abrogated by concomitant administration of the PI3 kinase inhibitor LY294002 (45 ± 3% vs. 50 ± 3%, ns). In congruence with reduction of infarct size rhCCN2 also improved recovery of left ventricular developed pressure (p < 0.05). Western blot analyses of extracts of ex vivo-perfused murine hearts also revealed that rhCCN2 evoked concentration-dependent increase of cardiac phospho-GSK3ß (serine-9) contents. CONCLUSIONS AND IMPLICATIONS: We demonstrate that post-ischemic administration of rhCCN2 increases the tolerance of ex vivo-perfused murine hearts to IRI. Mechanistically, this postconditioning effect of rhCCN2 appeared to be mediated by activation of the reperfusion injury salvage kinase pathway as demonstrated by sensitivity to PI3 kinase inhibition and increased CCN2-induced phosphorylation of GSK3ß (Ser-9). Thus, the rationale for testing rhCCN2-mediated post-ischemic conditioning of the heart in more complex models is established.

CCN2 exerts direct cytoprotective actions in adult cardiac myocytes by activation of the PI3-kinase/Akt/GSK-3ß signaling pathway.

We recently reported that transgenic mice with cardiac-restricted overexpression of CCN2/CTGF have substantially increased tolerance towards ischemia/reperfusion injury. The purpose of this study was to investigate to what extent fully differentiated cardiac myocytes are direct targets of CCN2, and to resolve the signaling mechanisms that convey the cardioprotective actions of CCN2. Akt and GSK-3ß were identified as putative intermediaries of intracellular signaling stimulated by recombinant human CCN2 (rhCCN2). Concentration-effect experiments revealed CCN2-stimulated phosphorylation of Akt (Ser473) and downstream GSK-3ß (Ser9) with EC50 ~250 nmol/L. CCN2-stimulated phosphorylation of Akt and GSK-3ß was sensitive to inhibition of PI3-kinase (LY294002). Phosphorylation of GSK-3ß was also sensitive to Akt-inhibition (API-2), demonstrating CCN2-engendered activation of a PI3-kinase/Akt/GSK-3ß-signaling pathway. A C-terminal peptide fragment of CCN2 (11.2 kD) displayed partial agonist activity, while two short peptides derived from the Thrombospondin- and the IGFBP- homology domains of CCN2, respectively, additively inhibited rhCCN2-stimulated Akt-phosphorylation. The viability of cardiac myocytes subjected to hypoxia/reoxygenation injury or doxorubicin-induced oxidative stress was assessed by assays of adenylate kinase and lactate dehydrogenase released from dying cells. Cardiac myocytes exposed to CCN2 displayed increased tolerance towards hypoxia/reoxygenation and doxorubicin-induced oxidative stress, an effect that was abrogated by inhibition of PI3-kinase. The cytoprotective actions of CCN2 reflected in the transcriptome of CCN2-stimulated cardiac myocytes (anti-apoptosis, stress, and wound-response gene programs). In conclusion, this study discloses the novel findings that cardiac myocytes are CCN2 target cells in which CCN2 increases tolerance towards hypoxia and oxidative stress via PI3-kinase-dependent Akt/GSK-3ß signaling.

Phadiatop infant in the diagnosis of atopy in children with allergy-like symptoms.

Background and Objective. Allergy-like symptoms such as wheezing and eczema are common in young children and an early diagnosis is important to initiate correct management. The objective of this study was to evaluate the diagnostic performance of Phadiatop Infant, an in vitro test for determination of early sensitisation to food and inhalant allergens. Patients and Methods. The study was conducted, retrospectively, using frozen sera from 122 children (median age 2.7 years) admitted to the hospital with suspected allergic symptoms. The doctor's diagnosis atopic/nonatopic was based on routinely used procedures such as clinical evaluation, SPT, total and allergen-specific IgE antibodies. The performance of Phadiatop Infant was evaluated in a blinded manner against this diagnosis. Results. Eighty-four of the 86 children classified as atopic showed a positive Phadiatop Infant test. Thirty-six were classified as nonatopic, 32 of who had a negative test. With a prevalence of atopy of 70% in this population, this gives a sensitivity of 98%, a specificity of 89%, and a positive and negative predictive value of 95% and 94%, respectively. Conclusion. The results from the present study suggest that Phadiatop Infant could be recommended as a complement to the clinical information in the differential diagnosis on IgE-mediated disease in young children with allergy-like symptoms.