Receptors & Clinical Investigation (E-Journal - Smart Science & Technology)
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Spry2 is a novel therapeutic target for periodontal tissue regeneration through fibroblast growth factor receptor signaling and epidermal growth factor signaling
Get PDFSprouty2 (Spry2) inhibits the activation of the extracellular signal-regulated kinase (ERK) pathway via receptor tyrosine kinase signaling. In a recent paper published in Journal of Cellular Biochemistry, we demonstrated that transfection of a dominant-negative mutant of Spry2 enhanced fibroblast growth factor (FGF)- and epidermal growth factor (EGF)-induced ERK activation in osteoblasts. In contrast, it decreased their activation in gingival epithelial cells. Consistent with these observations, the sequestration of Spry2 increased osteoblast proliferation by FGFR and EGFR stimulation, whereas it decreased gingival epithelial cell proliferation via the ubiquitination and degradation of EGF receptors (EGFR). In addition, reduction of Spry2 activity upregulated Runx2 expression and downregulated Twist, a negative regulator of Runx2 through FGFR and EGFR signaling, resulting in enhanced osteoblastogenesis in osteoblasts. Furthermore, we also found that suppression of Spry2 upregulated cell proliferation and migration in human periodontal ligament cell lines when they were stimulated by both FGF and EGF, and led to a shift in macrophage polarization, exerted immunosuppressive and tissue-repairing effects in macrophages. These results suggest that the application of a Spry2 inhibitor may effectively resolve inflammation by periodontitis and allow periodontal ligament and alveolar bone to grow and block the ingrowth of gingival epithelial cells in bony defects, biologically mimicking the barrier effect seen in conventional GTR. This approach has potential for developing a new regeneration strategy
Membrane Stretch and Angiotensin II type 1a Receptor: Causes and Role in the Myogenic Response
Get PDFThe ligand-independent activation of Angiotensin II type 1 receptors following vascular stretch plays very important (patho) physiological roles. Indeed, recent studies have implicated this mechanism in cardiac hypertrophy under conditions of pressure overload and it has shown to be indispensable in the regulation of the myogenic response in smooth muscle cells of small resistance arteries, as well as mesenteric and renal resistance arteries. The information discussed in here will highlight the involvement of the mechanoactivation of the Angiotensin II type I receptors in the development of the myogenic response and the molecular mechanisms modulating them following activation
Competitive and compensatory effects of androgen signaling and glucocorticoid signaling
Get PDFAndrogens and glucocorticoids have competitive and compensatory effects in several physiological and pathophysiological processes. Although blood androgen levels affect blood glucocorticoid levels and vice versa, it does not fully explain the relationship between the effects of androgens and glucocorticoids. Androgens and glucocorticoids exert their functions through binding to androgen receptor (AR) and glucocorticoid receptor (GR), respectively. AR homodimer and GR homodimer bind to the androgen response element (ARE) and glucocorticoid response element (GRE), respectively, where they positively or negatively regulate transcription. AR/GR heterodimer can also form but whether it has a physiological role is unclear. Notably, some ARE/GRE sites are recognized by both AR and GR. This review focuses on the functional interventions between androgen signaling and glucocorticoid signaling in target cells that are involved in muscle atrophy, lipid metabolism in adipocytes and hepatocytes, and pancreatic ?-cell death. Androgens and glucocorticoids exert opposite effects by differentially regulating key genes (e.g., insulin-like growth factor-1, atrogin-1, and thioredoxin-interacting protein) involved in these physiological processes. We also review functional compensation between these steroids in the development of castration-resistant prostate cancer in which glucocorticoids compensate for the castration-induced loss of AR function by activating key genes (e.g., serum/glucocorticoid-regulated kinase 1). The gene expressions regulated by androgens and glucocorticoids are regulated through at least three different mechanisms in target cells: (i) regulation of applicable ligand levels by modulation of steroid metabolite enzyme levels, (ii) regulation of each other\u27s receptor levels, and (iii) competitive binding between AR and GR on ARE/GRE sites. Recent findings shed light on the complicated relationship between androgen signaling and glucocorticoid signaling in various cellular processes
A family of sterol sensors/transporters at membrane contact sites: Regulation of ORP-VAP complexes by sterol ligands
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Androgen receptor and intracrine androgen signaling in endometrial carcinomas
Get PDFThe androgen receptor (AR) is known to play critical roles in the malignancy of prostate cancer as well as the management of male reproductive organs. Endometrial carcinoma, one of the major female cancers, is considered an androgen-related cancer. However, the importance of androgen signaling through its receptor in endometrial carcinomas has not yet been clarified. We recently demonstrated the significance of androgen signaling and intracrine dihydrotestosterone (DHT) in endometrial carcinomas as follows: 1) A positive status of androgen receptor (AR) was significantly associated with high rates of progression-free survival (PFS), but not with endometrial cancer-specific survival (ECSS) in endometrial carcinoma patients; 2) The potent androgen DHT was synthesized from testosterone by 5?-reductase in endometrial carcinoma tissues; and 3) endometrial carcinoma patients that were AR/5?-reductase type 1 double-negative had significantly worse PFS and ECSS. These findings suggest that androgen signaling exerts anti-cancer effects through the intratumoral DHT-AR pathway in endometrial carcinomas. In this highlight article, we describe androgen signaling in endometrial carcinomas, focusing mainly on our recent study entitled “The role of 5?-reductase type 1 associated with intratumoral dihydrotestosterone concentrations in human endometrial carcinoma” and discuss the findings of some previous related studies
Baby Steps Toward Modelling The Full human Programmed Death-1 (PD-1) Pathway
Get PDFImmune checkpoints play a vital role in regulating the immune system. They preserve the immunological balance between preventing continuous activated immune responses and defending against chronic infections and cancer. Blocking the immune inhibitory checkpoints pathways recently emerged as a ‘game changer’ approach in cancer and antiviral immunotherapy. Modeling these pathways at the atomic level provides a key step toward rationally designing selective blockers for these pathways. Current crystal structures for the immune checkpoints are mainly not for human and are very limited in their scope of interactions. Our team has been focused on building atomistic models for these proteins, characterizing their protein-protein interactions and designing new inhibitory drugs for their activity. This article highlights our recent study on modelling the human Programmed Death-1 (hPD-1) pathway by characterizing the interactions between hPD-1 and its two human ligands. In this study, we showed that hPD1 binds differently to its two ligands. We also showed that the modes of binding for each ligand are different between mouse and human, emphasizing the limited information in current mouse crystal structures. Our findings enhanced the understanding of the receptor-ligand(s) interactions and formed a significant step toward building a full model for the whole PD1 pathway. This undoubtedly will foster the ongoing efforts to develop antibodies and small molecule drugs against this important T cell immune-regulatory mechanism
Identification of Acanthamoeba Membrane Protein That is Recognized by TLR4 on Corneal Epithelial Cells
Get PDFWe have shown that Acanthamoeba spp. activate TLR4 on corneal epithelial cells and induce secretion of chemokines. However, the components of Acanthamoeba trophozoites that induce chemokines production remain unknown. We sought to identify the trophozoite molecules that interact with TLR4 on human corneal epithelial (HCE) cells and trigger IL-8 production. Acanthamoeba membrane protein (AcMP) was isolated by homogenization of trophozoites. The supernatants were collected, solubilized, and membrane fractions were separated by centrifugation using Mem-PERTM plus kit. To examine functional activity of AcMP, HCE and TLR4-expressing HEK293 cells were incubated with or without A. castellanii (1×105 cells/ml) and AcMP (10, 25, and 50 µg/ml) for 24 hours. AcMP was chromatographed by fast protein liquid chromatography (FPLC) and fractions were pooled into four peaks (AcMP-P1 - AcMP-P4). TLR4-ligand in AcMP-P1 - AcMP-P4 was determined by Western blotting. HEK293 and HCE cells were incubated with or without A. castellanii, lipopolysaccharide (LPS, 10 µg/ml), and AcMP-P1 - AcMP-P4 (20 µg/ml) for 24 hours. qRT-PCR and ELISA were used to examine the ability of AcMP-P1 - AcMP-P4 to stimulate IL-8 production in HEK293 and HCE cells. Inhibition of TLR4 involved preincubating HEK293 and HCE cells for 1 hour with neutralizing TLR4-antibody (10 µg/ml) or with the control antibody (10 µg/ml, goat serum) followed by incubation with or without A. castellanii, LPS, and AcMP-P2 for 24 hours. AcMP induced significant IL-8 production at doses of 10, 25, and 50 µg/ml in HEK293 cells while IL-8 mRNA expression and IL-8 secretion were significantly increased in HCE cells at the dose of 50 µg/ml. Treatments of HEK293 with FPLC chromatographed trophozoites’ proteins, AcMP-P1 - AcMP-P4; only AcMP-P2 upregulated significant IL-8 production and mRNA expression. Western blotting of AcMP-P1 - AcMP-P4 showed TLR4-antigen in AcMP-P2 and was recognized an approximate 15-kDa protein band. Anti-TLR4 antibody attenuated IL-8 secretion that is stimulated by AcMP-P2 from HEK293 and HCE cells. These results suggest that A. castellanii trophozoites recognize TLR4 on HCE and HEK293 cells by an approximate 15-kDa molecular mass protein of AcMP and induce IL-8 secretion
Structural Characterization of Modification on the Interface between a Ligand and its Receptor for Biopharmaceuticals
Get PDFThe binding of a protein to its target is a major mode of action for most biopharmaceutical therapies with over 70% of biopharmaceuticals involved in binding between the protein and its target. The interfaces between a biopharmaceutical and its target are key regions for its efficacy. Any modifications to the amino acids at the interfaces invariably affect interactions between the biopharmaceutical and its receptor and may result in lowering therapeutic efficacy. Degradations of biopharmaceuticals by asparagine (Asn) deamidation and/or aspartate (Asp) isomerization have been well characterized and those modifications at the interfaces have resulted in a loss of activity. To characterize modification hot-spots on the interfaces, it is necessary to identify the amino acid residues on the interfaces. We recently addressed a visualization tool for amino acids on the interfaces between a protein ligand and its receptor. This tool was applied to visualize ligand protein-receptor interaction and antigen-antibody interaction. As a model system for ligand protein-receptor interaction, erythropoietin (EPO) and its receptor were selected and amino acids on the interfaces were identified. Modifications on the interfaces were then investigated. Deamidation of Asn was identified at two amino acid residues, Asn47 and Asp147, on Interface 1 of EPO. The relative contents of deamidated residues on the interface of EPO were in the range of 3-5% of the total. As a model system for antigen-antibody interaction, Herceptin and its receptor, HER2, were chosen and amino acids on the interfaces were identified. Then modifications on the interfaces were assessed. Deamidation of the light chain Asn30 and heavy chain Asn55 were identified. The relative contents of the deamidated residues on the interfaces were in the range of 8-9% of the total. Along with deamidation, another modification, isomerization, was identified at the amino acid residue Asp102 of the heavy chain, and the level of oxidation was 13.5% of the total. Our studies provide a targeted method focusing on the interface between a protein and its target that can be coupled with other applications, for example, identification of modified amino acids on the interfaces
A Potential Role of SDF-1/CXCR4 Chemotactic Pathway in Wound Healing and Hypertrophic Scar Formation
Get PDFFibroproliferative disorders are an ongoing clinical issue that is prevalent within society today. These disorders generally manifest themselves by an overproduction of fibrotic tissue with unknown provocation resulting in numerous detrimental defects. Cellular migration of blood-borne cells via the chemotactic pathway, consisting of stromal cell-derived factor 1 and its receptor, CXCR4, has been strongly implicated in post-burn hypertrophic scar formation. Evidence has shown this pathway has potential as a therapeutic target in the formation of hypertrophic scar and likely in other fibroproliferative disorders
IL-10: Expanding the Immune Oncology Horizon
Get PDFRecent advances in immunoncology have dramatically changed the treatment options available to cancer patients. However, the fundamental challenges with this therapeutic modality are not new and still persist with the current wave of immunoncology compounds. These challenges are centered on the activation and expansion, induction of intratumoral infiltration and persistence of highly activated, cytotoxic, tumor antigen specific CD8+ T cells. We have investigated the anti-tumor mechanism of action of pegylated recombinant interleukin-10, (PEG-rIL-10) both pre-clinically with murine (PEG-rMuIL-10) and now clinically (AM0010) with human pegylated interleukin-10. The preponderance of data suggest that IL-10’s engagement of its receptor on CD8+ T cells enhances their activation status leading to antigen specific expansion. Quantitation of CD8+ T cell tumor infiltration reveals that treatment of both humans and mice with pegylated rIL-10 results in 3-4 fold increases of intratumoral, cytotoxic, CD8+ T cells. In addition, mice cured of their tumors with PEG-rMuIL-10 exhibit long term immunological protection from tumor re-challenge and long term treatment of cancer patients with AM0010 results in the persistence of highly activated CD8+ T cells. Cumulatively, these data suggest the IL-10 represents an emerging therapeutic that specifically addresses the fundamental challenges of the current wave of immunoncology assets