Induce Cell Signaling Research Articles

Development of a top-down MS assay for specific identification of human periostin isoforms.

Periostin is a matricellular protein encoded by the POSTN gene that is alternatively spliced to produce ten different periostin isoforms with molecular weights ranging from 78 to 91kDa. It is known to promote fibrillogenesis, organize the extracellular matrix, and bind integrin-receptors to induce cell signaling. As well as being a key component of the wound healing process, it is also known to participate in the pathogenesis of different diseases including atopic dermatitis, asthma, and cancer. In both health and disease, the functions of the different periostin isoforms are largely unknown. The ability to precisely determine the isoform profile of a given human sample is fundamental for characterizing their functional significance. Identification of periostin isoforms is most often carried out at the transcriptional level using RT-PCR based approaches, but due to high sequence homogeneity, identification on the protein level has always been challenging. Top-down proteomics, where whole proteins are measured by mass spectrometry, offers a fast and reliable method for isoform identification. Here we present a fully developed top-down mass spectrometry assay for the characterization of periostin splice isoforms at the protein level.

Molecular analysis of the extracellular microenvironment: from form to function.

The extracellular matrix (ECM) proteome represents an important component of the tissue microenvironment that controls chemical flux and induces cell signaling through encoded structure. The analysis of the ECM represents an analyticalchallenge throughhigh levels of post-translational modifications, protease-resistant structures, and crosslinked, insoluble proteins. This review provides a comprehensive overview of the analytical challenges involved in addressing the complexities of spatially profiling the extracellular matrix proteome. A synopsis of the process of synthesizing the ECM structure, detailing inherent chemical complexity, is included to present the scope of the analytical challenge. Current chromatographic and spatial techniques addressing these challenges are detailed. Capabilities for multimodal multiplexing with cellular populations are discussed with a perspective on developing a holistic view of disease processes that includes both the cellular and extracellular microenvironment.

Abstract B025: IL6 signaling in pre-malignant obese endometrium and endometrial cancer

Abstract Objective: Obesity associated chronic inflammatory status plays a role in the increased endometrial cancer incidences, IL-6 is one of those inflammatory molecules. To explore the potential of targeting pro-inflammation factors for endometrial cancer prevention, using multi-platform and multi-model analysis, we investigated IL6 and its associated signaling in pre-malignant endometrium and low-grade endometrial cancer. Methods: Illumina microarray and Nanostring nCounter® Pancancer Immune profiling panel gene expression profiling was performed on pre-malignant obese endometrium from diet induced obese Wistar rats, and endometrium from obese patients, lean endometrium was used as control. Clariom D assay was performed on RNA extracted from the gland and stroma of human obese endometrial carcinoma (OEC) and obese normal endometrium (ONE). IPA, nSolver, Transcriptome Analysis Console (TAC), and CIBERSORTx were used for data analysis. Ishikawa cells were 2D and 3D cultured, or 3D co-cultured with endometrial stroma cell line SHT290. Western blot was used for testing cell signal activity. Results: Pronounced IL-6 signaling (Z-score:2.3333, p=2.48E-02) was shown in pre-malignant obese endometrium of diet induced obese rats compared with lean littermates: NF-kB signaling (Z-score:2.714, p=3.47E-02), STAT3 signaling (Z-score:2.53, p=6.56E-04), and tumor microenvironment pathway (Z-score: 3.441, p=6.81E-05) were enhanced; PTEN signaling (Z-score:-1.997, p=2.76E-03) was attenuated. IL6R (FC=1.64, p=0.017), MMP2 (FC=1.938, p=0.026), IL1R2 (FC=1.75, p<0.0001), and CCL2 (FC=2.251, p=0.002) expression were increased. IL6 induced stronger STAT3 signaling, and weaker activation of MAPK signaling in Ishikawa cells 3D co-cultured with stroma cells, compared with 3D cultured Ishikawa cells without stroma cells. No IL-6 induced activation of MAPK signaling was observed with activation of Stat3 signaling in 2D-cultured Ishikawa cells. In pre-malignant obese endometrium from patients with high serum IL-6 levels, attenuated pro-inflammation signaling was seen compared with that from patients with low serum IL6 level: expression of IL1A (FC=-2.21, p=0.001), IRF8 (FC=-1.83, p=0.012), GAGE1 (FC=-3.49, p=0.028), and LBP (FC=-2.03, p=0.038) were decreased. Compared with pre-malignant obese endometrium, more regulatory T cell (Tregs, 0.1276 vs 0.077, p=0.04), and less eosinophils (0.003 vs 0.022, p=0.028) in the stroma, and more neutrophils (0.0145 vs. 0, p=0.02) in the tumor glands were observed in obese EC, implicating immuno-suppression in OEC. Conclusion: Due to the elevated systemic pro-inflammation status, pre-malignant obese endometrium demonstrated activated IL-6 signaling. IL-6 induced cell signaling varied by cellular microenvironment. In pre-malignant endometrium from obese women with high systemic IL-6 level, attenuated inflammation signaling was observed compared to those with low serum IL6 levels. Immuno-suppression was implicated in obese EC. Citation Format: Qian Zhang, Brenda Melendez, Xiaoping Su, Mikayla Bowen, Joseph Celestino, Melinda Sue Yates, Karen Lu. IL6 signaling in pre-malignant obese endometrium and endometrial cancer [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr B025.

Axon-derived PACSIN1 binds to the Schwann cell survival receptor, LRP1, and transactivates TrkC to promote gliatrophic activities.

Schwann cells (SCs) undergo phenotypic transformation and then orchestrate nerve repair following PNS injury. The ligands and receptors that activate and sustain SC transformation remain incompletely understood. Proteins released by injured axons represent important candidates for activating the SC Repair Program. The low-density lipoprotein receptor-related protein-1 (LRP1) is acutely up-regulated in SCs in response to injury, activating c-Jun, and promoting SC survival. To identify novel LRP1 ligands released in PNS injury, we applied a discovery-based approach in which extracellular proteins in the injured nerve were captured using Fc-fusion proteins containing the ligand-binding motifs of LRP1 (CCR2 and CCR4). An intracellular neuron-specific protein, Protein Kinase C and Casein Kinase Substrate in Neurons (PACSIN1) was identified and validated as an LRP1 ligand. Recombinant PACSIN1 activated c-Jun and ERK1/2 in cultured SCs. Silencing Lrp1 or inhibiting the LRP1 cell-signaling co-receptor, the NMDA-R, blocked the effects of PACSIN1 on c-Jun and ERK1/2 phosphorylation. Intraneural injection of PACSIN1 into crush-injured sciatic nerves activated c-Jun in wild-type mice, but not in mice in which Lrp1 is conditionally deleted in SCs. Transcriptome profiling of SCs revealed that PACSIN1 mediates gene expression events consistent with transformation to the repair phenotype. PACSIN1 promoted SC migration and viability following the TNFα challenge. When Src family kinases were pharmacologically inhibited or the receptor tyrosine kinase, TrkC, was genetically silenced or pharmacologically inhibited, PACSIN1 failed to induce cell signaling and prevent SC death. Collectively, these studies demonstrate that PACSIN1 is a novel axon-derived LRP1 ligand that activates SC repair signaling by transactivating TrkC.

Retinoic Acid Induced Cell Signaling as a Counter Against Disease

Many disease processes result from disruption of physiologic cell signaling pathways. Cancer often develops from the loss of cell cycle regulation, while inflammatory disease results from dysregulated immune activity. Likewise, many microbial infections avoid immune clearance by interfering with cellular antimicrobial pathways. Retinoic Acid (RA) is a dynamic compound, derived from vitamin A, that can regulate various signaling pathways. RA induced cell signaling has proven beneficial against different diseases, such as Acute Promyelocytic Leukemia (APL) and psoriasis. Against APL, RA induces cellular differentiation in cancer cells to restore proper function. In psoriasis, RA downregulates inflammatory pathways, such as NF-κB. RA’s anti-inflammatory properties have also been examined in the context of sepsis, where recent animal studies have shown positive benefits. Along with regulating inflammation, RA exhibits indirect antimicrobial properties. Unlike conventional antimicrobials which target pathogens directly, RA functions as a host-directed therapy (HDT), promoting cell antimicrobial defenses. Recent studies examining RA have shown that it can improve macrophage clearance of microbial pathogens and stimulate the antiviral type-I interferon (IFN) response. RA’s effectiveness has been demonstrated against clinically relevant pathogens, such as Mycobacterium tuberculosis, Aspergillus fumigatus, and Measles virus. In this review, the therapeutic potential of RA to treat various diseases by regulating cell signaling pathways will be explored.

Modeling an ascending infection by Ureaplasma parvum and its cell signaling and inflammatory response at the feto-maternal interface.

Ascending bacterial infection is associated with ∼ 40% of spontaneous preterm birth (PTB), and Ureaplasma spp. is one of the most common bacteria isolated from the amniotic fluid. Developing novel in vitro models that mimic in vivo uterine physiology is essential to study microbial pathogenesis. We utilized the feto-maternal interface organ-on-chip (FMi-OOC) device and determined the propagation of Ureaplasma parvum, and its impact on cell signaling and inflammation. FMi-OOC is a microphysiologic device mimicking fetal membrane/decidua interconnected through microchannels. The impact of resident decidual CD45+ leukocytes was also determined by incorporating them into the decidual chamber in different combinations with U. parvum. We tested the propagation of live U. parvum from the decidual to the amniochorion membranes (immunocytochemistry and quantitative PCR), determined its impact on cytotoxicity (LDH assay), cell signaling (JESSTM Western Blot), cellular transition (immunostaining for vimentin and cytokeratin), and inflammation (cytokine bead array). U. parvum transversed the chorion and reached the amnion epithelium after 72hours but did not induce cell signaling kinases (p38MAPK and JNK) activation, or cellular transition (epithelial-mesenchymal), regardless of the presence of immune cells. The inflammatory response was limited to the choriodecidual interface and did not promote inflammation in the amnion layer. Our data suggest that U. parvum is poorly immunogenic and does not produce massive inflammatory changes at the feto-maternal interface. We speculate that the presence of U. parvum may still compromise the feto-maternal interface making it susceptible to other pathogenic infection.

Role of ST6GAL1 and ST6GAL2 in subversion of cellular signaling during enteroaggregative Escherichia coli infection of human intestinal epithelial cell lines

Emerging evidence have suggested that aberrant sialylation on cell-surface carbohydrate architecture may influence host-pathogen interactions. The α2,6-sialyltransferase (ST) enzymes were found to alter the glycosylation pattern of the pathogen-infected host cell-surface proteins, which could facilitate its invasion. In this study, we assessed the role of specific α2,6-ST enzymes in the regulation of enteroaggregative E. coli (EAEC)-induced cell signaling pathways in human intestinal epithelial cells. EAEC-induced expression of α2,6-ST family genes in HCT-15 and INT-407 cell lines was assessed at mRNA level by qRT-PCR. Specific esi-RNA was used to silence the target ST-gene in each of the EAEC-infected cell type. Subsequently, the role of these enzymes in regulation of EAEC-induced cell signaling pathways was unraveled by analyzing the expression of MAPkinases (ERK1/2, p38, JNK) and transcription factors (NFκB, cJun, cFos, STAT) at mRNA and protein levels by qRT-PCR and western immunoblotting, respectively, expression of selected sialoglycoproteins by western immunoblotting along with the secretory IL-8 response using sandwich ELISA. ST6GAL-1 and ST6GAL-2 were efficiently silenced in EAEC-infected HCT-15 and INT-407 cells, respectively. Significant reduction in EAEC-induced activation of MAPKs, transcription factors, sialoglycoproteins, and IL-8 secretion was noted in ST-silenced cells in comparison to the respective control cells. We propose that ST6GAL-1 and ST6GAL-2 are quintessential for EAEC-induced stimulation of MAPK-mediated pathways, resulting in activation of transcription factors, leading to an inflammatory response in the human intestinal epithelial cells. Our study may be helpful to design better therapeutic strategies to control EAEC- infection. KEY POINTS: • EAEC induces α2,6-sialyltransferase (ST) upregulation in intestinal epithelial cells • Target STs (ST6GAL-1 & ST6GAL-2) were efficiently silenced using specific esiRNAs • Expression of MAPKs, transcription factors & IL-8 was reduced in ST silenced cells.

Roles of ERK/JNK in carbon black induced AP-1 cell signaling pathway changes

To investigate the role of ERK/JNK in the alteration of activator protein-1(AP-1) signaling pathway in human embryonic lung fibroblasts(HELFs) induced by carbon black. HELFs were cultured in RPMI 1640 medium containing 0, 15, 30, 60, 120 or 240 μg/mL carbon black for 24 h, and the appropriate dose of carbon black was determined by MTT assay result. HELFs were divided into three groups: HELFs, HELFs transfected with ERK dominant negative mutant plasmid(DN-ERK) and HELFs transfected with JNK dominant negative mutant plasmid(DN-JNK). 100 μg/mL carbon black was used to treat HELFs(CB), DN-ERK HELFs(CB-DN-ERK), DN-JNK HELFs(CB-DN-JNK), and HELFs without any treatment were considered as control group. At 0, 1, 2, 4, 8, 12, 24 and 36 h of CB and control groups HELFs, the western blot was used to detect ERK, p-ERK, JNK, p-JNK, p38, p-p38, c-Jun, p-c-Jun, c-Fos, p-c-Fos protein expression levels, and AP-1 activity was detected by luciferase method. Whereas CB-DN-ERK and CB-DN-JNK HELFs were detected only at 24 h. Compared with the protein expression levels at 0 h, CB group HELFs ERK and p-ERK protein expression increased at each time point, whereas p38 protein expression decreased. AP-1 activity of CB group HELFs was declined to the lowest at 8 h(0.72±0.12), and upregulated to the peak at 36 h(1.38±0.11). CB group HELFs c-Fos, p-c-Fos and c-Jun protein expression levels at each time point from 1 h to 24 h were greater than those of 0 h, and p-c-Jun protein expression levels at 1 h, 2 h, 4 h, 8 h, 36 h were also greater than those of 0 h. CB group HELFs AP-1 activity, ERK, p-ERK, JNK, p-JNK, p38, p-p38, c-Jun, p-c-Jun, c-Fos, p-c-Fos protein expression levels changes followed biphasic patterns. There were no statistically significant differences in AP-1 activity between CB group HELFs(1.03±0.10) and CB-DN-ERK group(1.02±0.04) or CB-DN-JNK group(1.09±0.10) HELFs(t=0.16, P=0.88; t=0.73, P=0.50). However, compared with CB group HELFs, c-Fos(t=5.31, P=0.01), p-c-Fos(t=4.33, P=0.01), p-c-Jun(t=10.95, P& lt; 0.01)in CB-DN-JNK group, and c-Fos protein expression levels in CB-DN-ERK group(t=42.72, P& lt; 0.01)were significantly decreased. While carbon black induces HELFs increased protein expression levels of ERK, p-ERK, c-Jun, p-c-Jun, c-Fos and p-c-Fos, JNK may upregulate c-Fos, p-c-Fos, p-c-Jun protein expression levels, and ERK may upregulate c-Fos protein expression level.

Low TFPI Mice in Models of Atherosclerosis and Endotoxemia

Tissue factor (TF), an integral membrane protein, binds factor VIIa (FVIIa) and the FVIIa/TF catalytic complex is responsible for the initiation of coagulation and enhances the inflammatory response by inducing cell signaling through protease activated receptor-2. Tissue Factor Pathway Inhibitor (TFPI) is an important endogenous regulator that produces FXa-dependent feedback inhibition of the FVIIa/TF complex and directly inhibits FXa. Through alternative mRNA splicing, TFPI is expressed in several isoforms, which possess unique functional properties and are present at different locations. TFPIα contains three Kunitz-type protease inhibitory domains (K1, K2, K3) and a basic C-terminus. It is a soluble form that produces FVIIa/TF inhibition (through K1) and whose inhibition of FXa (through K2) is dramatically enhanced by protein S (PS, through K3 binding). TFPIβ is a GPI-anchored cell surface form (containing K1 and K2) that inhibits FVIIa/TF and inhibits FXa well in the absence of PS. TFPIγ is a soluble two Kunitz domain (K1, K2) form that inhibits FVIIa/TF, but inhibits FXa weakly. It is expressed in the mouse, but not in man.Our initial TFPI gene-disruption in mice targeted the K1 domain in all expressed TFPI isoforms. Due to alternative mRNA splicing, this disrupted gene continues to express forms of TFPI (at ~50% the normal plasma level) that lack the K1 domain (TFPIΔK1) and thus do not inhibit FVIIa/TF. TFPIK1(-/-) gene-disrupted mice die in utero of intravascular coagulation and a consumptive coagulopathy. We have rescued TFPIK1(-/-) mice using a Tie2-hTFPIα transgene that directs endothelial cell and hematopoietic cell expression of hTFPIα . Rescued mice reproduce and appear healthy. The level of hTFPIα in the plasma and blood platelets (at 7.5 x 108/mL) of Tie2-hTFPIαtransgene rescued TFPIK1(-/-) mice is 15.3 + 4.4 and 24.7 + 3.6 pM (at 7.5x108/mL platelets). These values are 1/4 and 1/3 the levels of mTFPIα in normal animals, respectively. Further, hTFPIα is at least 3-fold less active than mTFPIα in mouse plasma thrombin generation assays induced with mouse thromboplastin or mouse FXa. Therefore, we refer to these Tie2-hTFPIαtransgene rescued TFPIK1(-/-) mice as “Low TFPI mice.” All mice used in the studies below were in the C57Bl/6 (B6) genetic background.An earlier study had noted a modest, localized increase in atherosclerosis in 34 week old TFPIK1(+/-)/ApoE(-/-) mice fed a normal diet. For the study reported herein, Low TFPI/ApoE(-/-) and ApoE(-/-) mice were fed a high fat (25.2% by weight) diet from weaning. At 35 weeks of age, compared to the ApoE(-/-) mice the Low TFPI/ApoE(-/-) mice had developed significantly increased thrombin-antithrombin (TAT) and interleukin-6 (IL6) levels, but, surprisingly, reduced cholesterol and triglyceride levels, and no significant difference in atherosclerotic plaque area (see Table 1 below). Thrombi were not detected in tissues, including the innominate artery in either mouse strain.Injection of lipopolysaccharide (LPS) in mice has been used extensively in investigations of the innate immune response and of the role of coagulation-related proteins in acute endotoxemia. Low TFPI and control B6 mice respond similarly to a low, sub-lethal 2.5 mg/kg (IP) LPS challenge. Studies with increasing doses of LPS show Low TFPI mice are ~5 times more sensitive to LPS with respect to mortality (LD50 ~4 vs. 20 LPS mg/kg). At a 10 mg/kg LPS challenge, Low TFPI mice have significantly increased TAT, IL6 and INF-γ, and CXCL1 levels compared to B6 mice at 24 hours (see Table 2 below).I n summary, concomitant Low TFPI in fat-fed ApoE(-/-) mice was associated with increased procoagulant (TAT) and cytokine (IL-6) biomarkers, but no increase in atherosclerosis. The latter result was potentially confounded by reductions in cholesterol and triglycerides in the Low TFPI/ApoE(-/-) mice. Low TFPI mice show similar cytokine responses as WT mice to a low-dose, sub-lethal endotoxin challenge, but are markedly more sensitive to high-dose endotoxin challenge. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Complete TFPI Gene-Disruption Is More Severe Than TFPI Kunitz-1 Gene-Deletion in Mice

Tissue factor (TF), an integral membrane protein, binds factor VIIa (FVIIa) and the FVIIa/TF catalytic complex is responsible for the initiation of coagulation and enhances the inflammatory response by inducing cell signaling through protease activated receptor-2. Tissue Factor Pathway Inhibitor (TFPI) is an important endogenous regulator that produces FXa-dependent feedback inhibition of the FVIIa/TF complex and directly inhibits FXa. Through alternative mRNA splicing, TFPI is expressed in several isoforms, which possess unique functional properties and are present at different locations. TFPIα contains three Kunitz-type protease inhibitory domains (K1, K2, K3) and a basic C-terminus. It is a soluble form that produces FVIIa/TF inhibition (through K1) and whose inhibition of FXa (through K2) is dramatically enhanced by protein S (PS, through K3 binding). TFPIβ is a glycosyl phosphatidyl inositol (GPI)-anchored cell surface form (containing K1 and K2) that inhibits FVIIa/TF and inhibits FXa well in the absence of PS. TFPIγ is a soluble two Kunitz domain (K1, K2) form that inhibits FVIIa/TF, but inhibits FXa weakly. It is expressed in the mouse, but not in man.Our initial TFPI gene-disruption targeted deletion of the K1 domain exon and is referred to herein as TFPIK1(-). Due to alternative mRNA splicing, this disrupted gene continues to express forms of TFPI that lack the K1 domain (TFPIΔK1) and thus do not inhibit FVIIa/TF. TFPIK1(-/-) gene-disrupted mice die in utero of intravascular coagulation and a consumptive coagulopathy. Others have shown that TFPIK1(-/-) mice can be rescued through gestation by concomitant FVII deficiency, but they succumb to perinatal consumptive coagulopathy. TFPIK1(-/-) mice are also rescued by low levels of TF, and by deletion of protease-activated receptor-4 (PAR4), a mouse platelet thrombin receptor.Referred to herein as TFPI(-), mice with complete TFPI gene-disruption and devoid of protein expression were generated by deletion of the TFPI exons encoding the signal peptide plus N-terminal peptide and the K1 domain. A western blot of plasma from WT, TFPIK1(+/-), and TFPI(+/-) mice demonstrates a ~50% reduction in normal TFPI in heterozygous animals and the presence of TFPIΔK1 protein in TFPIK1(+/-) plasma, which is absent in TFPI(+/-) mice.The rescue efficiencies of TFPIK1(-/-) and TFPI(-/-) mice at weaning by transgenes Tie2-hTFPIαor Tie2-hTFPIβ which direct endothelial cell and hematopoietic cell expression of low levels of hTFPIαor hTFPIβrespectively, and Tf-mXK1-halb, in which the transferrin promoter (Tf) directs expression of mXK1-halb in the liver are shown in the table below. mXK1-halb is a chimera of the mouse factor X light chain, mouse TFPI K1 and human albumin, designed as a species-select, extended half-life, direct FVIIa/TF inhibitor. The plasma level of mXK1-halb in the Tf-mXK1-halb rescued TFPIK1(-/-) mice is 1.6+0.3 nM, demonstrating that the mXK1-halb chimeric protein possesses significant anti-FVIIa/TF activity. The most striking finding is that the mXK1-halb rescues all of the TFPIK1(-/-) mice and none of the TFPI(-/-) mice. Further, whereas PAR4 gene-deletion reportedly rescues 40% of the TFPIK1(-/-) mice, we have found that it rescues none of the TFPI(-/-) mice.Timed matings show that 62.5% of TFPIK1(-/-) embryos survive to embryonic day 11.5 (E11.5), but that TFPI(-/-) mice do not progress beyond E10.0. Treatment of pregnant dams with rivaroxaban by gavage improved survival of TFPI(-/-) embryos at E11.5 from 0 to 46%. Experiments to define the cause of death in the TFPI(-/-) embryos are in progress.The disparity between the rescue of TFPIK1(-/-) and TFPI(-/-) mice demonstrates a functionally significant in vivo activity for the TFPI Δ K1 proteins in TFPIK1(-/-) mice. This activity is likely directed against FXa or, possibly, some other moiety that is also affected by rivaroxaban. The residual activity of the TF PIΔK 1 proteins confounds the interpretation of previous studies using mice with the TFPI K1 (-) disrupted gene. Extrapolation of results in TFPIK1(-) mice to the TFPI(-) condition is inappropriate. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Targeting Drugs Against Fibroblast Growth Factor(s)-Induced Cell Signaling.

The fibroblast growth factor (FGF) family is comprised of 23 highly regulated monomeric proteins that regulate a plethora of developmental and pathophysiological processes, including tissue repair, wound healing, angiogenesis, and embryonic development. Binding of FGF to fibroblast growth factor receptor (FGFR), a tyrosine kinase receptor, is facilitated by a glycosaminoglycan, heparin. Activated FGFRs phosphorylate the tyrosine kinase residues that mediate induction of downstream signaling pathways, such as RAS-MAPK, PI3K-AKT, PLCγ, and STAT. Dysregulation of the FGF/FGFR signaling occurs frequently in cancer due to gene amplification, FGF activating mutations, chromosomal rearrangements, integration, and oncogenic fusions. Aberrant FGFR signaling also affects organogenesis, embryonic development, tissue homeostasis, and has been associated with cell proliferation, angiogenesis, cancer, and other pathophysiological changes. This comprehensive review will discuss the biology, chemistry, and functions of FGFs, and its current applications toward wound healing, diabetes, repair and regeneration of tissues, and fatty liver diseases. In addition, specific aberrations in FGFR signaling and drugs that target FGFR and aid in mitigating various disorders, such as cancer, are also discussed in detail. Inhibitors of FGFR signaling are promising drugs in the treatment of several types of cancers. The clinical benefits of FGF/FGFR targeting therapies are impeded due to the activation of other RTK signaling mechanisms or due to the mutations that abolish the drug inhibitory activity on FGFR. Thus, the development of drugs with a different mechanism of action for FGF/FGFR targeting therapies is the recent focus of several preclinical and clinical studies.

The future of skin tightening: Mechanical or biological?

AbstractIntroductionNot too long ago, the concept of nonexcisional skin tightening was a field of dreams. Currently, the field of nonexcisional skin tightening is heavily device dependent. Though great strides have been made in both minimally invasive skin tightening and totally noninvasive devices, will these be in use for decades? Advances in biological research and development have been rapid. Consumer demand for regenerative solutions is high, despite the warnings from the Food and Drug Administration (FDA) noting that benefits are unproven and that instances of charlatan practice are high. While genetic modification of food is frowned upon, the use of biomarkers in medicine has become a standard of care.Energy based devicesCurrent devices that have retained value in the skin tightening arena include transcutaneous radiofrequency (RF) and microfocused ultrasound. Laser resurfacing can improve mild skin laxity but its best use is wrinkle removal and pigment and textural improvement. RF‐based subdermal tightening consistently achieves a higher measured skin surface contraction than alternatives such as ultrasound‐assisted or laser‐assisted liposuction. High‐intensity electromagnetic field (HIFEM) improves that framework that supports the overlying skin, as well as reducing diastasis recti. HIFEM can generate a 17.9% skin surface area contraction. A combination of transcutaneous RF and targeted pressure energy significantly reduces skin surface irregularities, cellulite, and striae while measurably thickening the dermis.Injectables and biologiocalsBiologically based entries into the field include mechanically processed adipose‐derived stem cells, nanofat, and exosomes. Intradermal injection of nanofat and topical application of exosomes following microneedling can "resurface" skin by reversing many age related changes. A recently approved collagense drug can correct skin surface depressions such as cellulite, deformities following liposuction, and other areas of fibrosis.ConclusionSurgical skin excision has largely been replaced with minimally invasive and noninvasive alternatives. While energy based devices still dominate this field, biologicals are rapidly gaining ground. Substances that induce cell signaling can target cell senescence. The future of skin tightening will include treatment options that offer tightening, smoothing, dermal thickness improvement, hydration, and overall skin quality improvement.

Polyphenols by Generating H2O2, Affect Cell Redox Signaling, Inhibit PTPs and Activate Nrf2 Axis for Adaptation and Cell Surviving: In Vitro, In Vivo and Human Health.

Human health benefits from different polyphenols molecules consumption in the diet, derived mainly by their common activities in the gastrointestinal tract and at the level of blood micro-capillary. In the stomach, intestine and colon, polyphenols act as reducing agents preventing lipid peroxidation, generation and absorption of AGEs/ALEs (advanced glycation end products/advanced lipid oxidation end products) and postprandial oxidative stress. The low absorption of polyphenols in blood does not support their activity as antioxidants and their mechanism of activity is not fully understood. The results are from in vitro, animal and human studies, detected by relevant oxidative stress markers. The review carries evidences that polyphenols, by generating H2O2 at nM concentration, exogenous to cells and organs, act as activators of signaling factors increasing cell Eustress. When polyphenols attain high concentration in the blood system, they generate H2O2 at µM concentration, acting as cytotoxic agents and Distress. Pre-treatment of cells or organisms with polyphenols, by generating H2O2 at low levels, inhibits cellular PTPs (protein tyrosine phosphatases), inducing cell signaling through transcription of the Nrf2 (nuclear factor erythroid 2-related factor 2) axis of adaptation and protection to oxidation stress. Polyphenols ingestion at the right amount and time during the meal acts synergistically at the level of the gastrointestinal tract (GIT) and blood system, for keeping the redox homeostasis in our organism and better balancing human health.

The Dipeptidyl Peptidase-4 Inhibitor Linagliptin Directly Enhances the Contractile Recovery of Mouse Hearts at a Concentration Equivalent to that Achieved with Standard Dosing in Humans.

Despite a similar mechanism of action underlying their glucose-lowering effects in type 2 diabetes, dipeptidyl peptidase-4 (DPP-4) inhibitors have diverse molecular structures, raising the prospect of agent-specific, glucose-independent actions. To explore the issue of possible DPP-4 inhibitor cardiac heterogeneity, we perfused different DPP-4 inhibitors to beating mouse hearts ex vivo, at concentrations equivalent to peak plasma levels achieved in humans with standard dosing. We studied male and female mice, young non-diabetic mice, and aged diabetic high fat diet-fed mice and observed that linagliptin enhanced recovery after ischemia-reperfusion, whereas sitagliptin, alogliptin, and saxagliptin did not. DPP-4 transcripts were not detected in adult mouse cardiomyocytes by RNA sequencing and the addition of linagliptin caused ≤0.2% of cardiomyocyte genes to be differentially expressed. In contrast, incubation of C166 endothelial cells with linagliptin induced cell signaling characterized by phosphorylation of Akt and endothelial nitric oxide synthase, whereas the nitric oxide (NO) donor, S-nitroso-N-acetylpenicillamine increased serine 16 phosphorylation of the calcium regulatory protein, phospholamban in cardiomyocytes. Furthermore, linagliptin increased cardiomyocyte cGMP when cells were co-cultured with C166 endothelial cells, but not when cardiomyocytes were cultured alone. Thus, at a concentration comparable to that achieved in patients, linagliptin has direct effects on mouse hearts. The effects of linagliptin on cardiomyocytes are likely to be either off-target or indirect, mediated through NO generation by the adjacent cardiac endothelium.

Acute Signaling Responses To Resistance Exercise In Previously Trained And Untrained Skeletal Muscle

It has been indicated that human skeletal muscle has a heightened sensitivity to exercise stimulus when it has been previously trained i.e. possess a “muscle memory”. While previous studies have been directed to the aspects of myonuclear content and epigenetic modifications, no previous study have explored the potential of a muscle memory concerning signaling responses related to acute resistance exercise. PURPOSE: The aim here was to study whether basal and acute resistance exercise induced cell signaling is influenced by previous strength training history in human skeletal muscle. METHODS: 19 training naïve women and men completed 10 weeks of hypertrophy inducing unilateral strength training followed by 20 weeks of detraining. Subsequently, an acute resistance exercise session involving leg press and knee extensions was performed alternated with both legs. Vastus lateralis biopsies taken at rest and 1 h post exercise in both the Control- and Memory-leg. Immunoblotting was used to assess total content and phosphorylation status of proteins in the mTORC1- and related pathways. RESULTS: Following detraining leg muscle hypertrophy had been reversed, but the Memory-leg was on average 19% stronger than the Control-leg. There were no differences between legs with regard to total protein content of all the signaling proteins analyzed. The phosphorylation of AMPKThr172 and eEF2Thr56 was 16%, respectively 21%, higher in the Memory-leg compared to the Control-leg at both time points. The effect on AMPKThr172 was attributed to changes in the women only, whereas the effect on eEF2Thr56 was present in both sexes. Exercise induced an increased phosphorylation of mTORSer2448 (26-36%), S6K1Thr389 (6- to 7-fold) and S6Ser235/236 (13- to 18-fold), that did not differ between the Control- and Memory-leg. In contrast, post exercise phosphorylation of 4E-BP1Thr46 and 4E-BP1Ser65 was 18%, respectively 31%, higher in the Memory-leg compared to the Control-leg. For 4E-BP1Ser65 the effects were attributed to changes in the male subjects only. CONCLUSION: In summary, we illustrate that both basal- and exercise induced cell signaling important for muscle adaptations to strength training can be altered by previous training history, and that some of the changes seem to be sex dependent.

464-P: Linagliptin Directly Enhances Cardiac Contractile Recovery through Nitric Oxide Mediated Endothelial–Cardiomyocyte Communication

Despite a similar mechanism of action underlying their glucose-lowering effects, dipeptidyl peptidase-4 (DPP-4) inhibitors have diverse molecular structures, raising the prospect of agent-specific, glucose-independent actions. This possibility is supported by heterogeneous heart failure outcomes in cardiovascular trials, although few head-to-head comparisons exist. To explore the possibility of DPP-4 inhibitor cardiac heterogeneity, we perfused mouse hearts with different DPP-4 inhibitors at concentrations equivalent to their peak plasma levels achieved in humans with standard dosing (Cmax). We studied male and female mice, young nondiabetic mice and aged diabetic high fat diet-fed mice and observed that linagliptin enhanced recovery after ischemia-reperfusion, whereas sitagliptin, alogliptin and saxagliptin did not. DPP-4 transcripts were not detected in adult mouse cardiomyocytes by RNA-sequencing and the addition of linagliptin caused ≤0.2% of cardiomyocyte genes to be differentially expressed. In contrast, incubation of C166 endothelial cells with linagliptin induced cell signaling characterized by phosphorylation of Akt and endothelial nitric oxide synthase, whereas the nitric oxide (NO) donor, S-nitroso-N-acetylpenicillamine increased serine 16 phosphorylation of the calcium regulatory protein, phospholamban in cardiomyocytes. Thus, at concentrations achieved with standard dosing in humans, linagliptin has direct effects on the recovery of mouse cardiac contractility after ischemia-reperfusion, most likely through NO-mediated endothelial-cardiomyocyte communication. The complex cardiac actions of NO (that depend on comorbidity, concurrent medications and disease setting, for instance) may contribute to both the discordance in DPP-4 inhibitor outcomes between preclinical and clinical observations and the heterogeneous outcomes observed in clinical trials to date. Disclosure V. Yerra: None. S. Batchu: None. Y. Liu: None. T. Klein: Employee; Self; Boehringer Ingelheim International GmbH. A. Advani: Research Support; Self; Boehringer Ingelheim International GmbH. Funding Boehringer Ingelheim

Circulating exosomes from human lung transplant recipients with respiratory viral infections contain nucleic acids potential to activate innate immune signaling (cGAS/STING and RIG-1 Pathways)

Abstract Exosomes are 50–200nm size vesicles released by cells into the body fluids. We reported the presence of circulating exosomes with lung self-antigens (SAgs) (Collagen-V,K-α Tubulin) and donor HLA in lung transplant recipients (LTxRs) undergoing rejection. Respiratory viral infections (RVI) is a known risk factor for development of chronic lung allograft dysfunction (CLAD) post lung transplant. We postulated immune mechanism by which RVI increases risk for CLAD is by induction of exosomes with SAgs containing viral DNA/RNA capable of activating innate immune signaling via DNA/RNA sensing. Exosomes were isolated using ultracentrifugation. Exosomal DNA and RNA were used to generate libraries using Kapa Biosystem’s kit. Illumina 2×150bp pair-end reads were checked on FastQC, aligned to human/viral genome from CHIP seeker Database. Role of exosomes inducing cell signaling in human airway epithelial cells (KCC266) and Hep2 were analyzed by incubating exosomes from LTxRs with RVI or stable. Viral nucleic acid sequences were higher in exosomes from LTxRs with RVI while comparison with human genome identified the presence of DNA sequences specific to defensins, GTPase, apoptotic cleavage, and NMDA receptor in exosomes from RVI. We observed upregulation of proteins associated with cGAS/STING and RIG1 in KCC266 and Hep2 cells (STING; 1.5, cGAS; 1.9, pIRF3; 2.5, pTBK; 3.1, MAVS; 1.7 and IFNβ; 3.8 fold respectively) incubated with exosomes from LTxRs with RVI. We conclude that LTxRs with RVI leads to induction of circulating exosomes having unique viral nucleic acid sequences capable of inducing signaling. This can lead to activation of innate immune signaling resulting in adaptive immune responses to viral and donor antigens resulting in CLAD.

Transmembrane Peptides as Inhibitors of Protein-Protein Interactions: An Efficient Strategy to Target Cancer Cells?

Cellular functions are regulated by extracellular signals such as hormones, neurotransmitters, matrix ligands, and other chemical or physical stimuli. Ligand binding on its transmembrane receptor induced cell signaling and the recruitment of several interacting partners to the plasma membrane. Nowadays, it is well-established that the transmembrane domain is not only an anchor of these receptors to the membrane, but it also plays a key role in receptor dimerization and activation. Indeed, interactions between transmembrane helices are associated with specific biological activity of the proteins as cell migration, proliferation, or differentiation. Overexpression or constitutive dimerization (due notably to mutations) of these transmembrane receptors are involved in several physiopathological contexts as cancers. The transmembrane domain of tyrosine kinase receptors as ErbB family proteins (implicated in several cancers as HER2 in breast cancer) or other receptors as Neuropilins has been described these last years as a target to inhibit their dimerization/activation using several strategies. In this review, we will focus on the strategy which consists in using peptides to disturb in a specific manner the interactions between transmembrane domains and the signaling pathways (induced by ligand binding) of these receptors involved in cancer. This approach can be extended to inhibit other transmembrane protein dimerization as neuraminidase-1 (the catalytic subunit of elastin receptor complex), Discoidin Domain Receptor 1 (a tyrosine kinase receptor activated by type I collagen) or G-protein coupled receptors (GPCRs) which are involved in cancer processes.

Induced Pluripotent Stem Cell‐Derived Neuroprogenitor Cells: A Model for Studying Cannabinoid Induced Cell Signaling

Chronic pain is a growing epidemic and public health issue that continue to affect the aging population in the US. Managing pain is particularly difficult with patients who suffer from cancer‐associated pain, neuropathic pain, as well as central pain states. These conditions are typically treated inadequately with opioids, antidepressants, and anticonvulsive drugs. There is growing evidence that cannabinoids natural products are beneficial for a range of clinical conditions, including pain and inflammations. The use of cannabinoids instead of highly regulated prescription drugs is fast approaching and the development of such cannabinoid drugs requires well planned medical trials as well as very stringent guidelines for contaminants such as heavy metals, pesticides, mycotoxins, etc. However, federal guidelines have not kept up with this emerging industry. Currently, each state is left to manage their own set of regulations, resulting in an industry that has great uncertainty and chaos as it attempts to satisfy the growing demand for its products. A low‐cost cellular model to study the neurological effects of cannabinoids is challenging. Here we show that Neuroprognitor cells (NPs) derived from induced pluripotent stem cells (iPSCs) can be used as a model for studying pathways associated with pain and cannabinoids. NPs derived from iPSCs can be differentiated into multiple lineages of neurons and glial cells. These cells have the Cannabinoid Receptor 1 (CB1) present which, when activated by cannabinoids, inhibits the inflammatory and pain stimulation of Cyclooxygenase 2 (COX2). We show expression and activation of CB1 in correlation to Cannabinoids treatment. Likewise we show how, in iPSC‐derived NPs Cannabinoid treatment leads to the inhibition of Cox‐2, therefore theoretically lessoning the potential for inflammation and pain.Support or Funding InformationOmni International, Inc935 Cobb Place BlvdKennesaw Ga 30144

Circulating Exosomes from Human Lung Transplant Recipients Having Respiratory Viral Infections Contain Nucleic Acids and Activate Signaling Pathways CGAS/STING and RIG-1

PurposeExosomes are membrane bound vesicles are released by cells into body fluids. Our laboratory demonstrated the presence of circulating exosomes with lung self-antigens (Collagen-V and K-α Tubulin) and donor HLA in lung transplant recipients (LTxRs) undergoing rejection. Since respiratory viral infections (RVI) is a risk factor for development of chronic lung allograft dysfunction (CLAD) post lung transplant, we postulated that RVI can lead to induction of exosomes with self-antigens containing viral DNA/RNA capable of activating innate immune signaling via cGAS/STING and RIG1 pathways, a mechanism leading to immune activation resulting in CLAD.MethodsExosomes were isolated using ultracentrifugation. Size (50-200nm) was determined using nanosight. DNA and RNA were isolated using kits and quantified on the Nanodrop. Libraries were generated using Kapa Biosystem's library kit. The raw Illumina 2x150bp pair-end reads were checked on FastQC and were aligned to the human and viral genome build from CHIPseeker Database. Validation was done using antibodies and primers for respiratory syncytial virus, coronavirus, and rhinovirus. To determine the role of exosomes to induce cell signaling and endoplasmic stress (ER), airway epithelial cells (KCC266), and Hep2 cells were incubated with exosomes from LTxRs with RVI or stable.ResultsViral nucleic acid sequences were noted in higher levels in exosomes from LTxRs with RVI in comparison to stable. Comparison with human genome identified the presence of DNA sequences specific to defensins, GTPase, apoptotic cleavage, and NMDA receptor in RVI LTxRs. Further, we identified upregulation of proteins associated with cGAS/STING and RIG1 (MAVS, MDA5, IFNβ) and ER stress (PERK, ATF4 and BiP) in KCC266 and Hep2 cells incubated with exosomes from LTxRs with RVI, but not stable. In contrast, exosomes from stable LTxRs had 91 sequences for MAP kinase and cell death signaling pathways.ConclusionWe conclude that LTxRs diagnosed with RVI leads to induction of circulating exosomes having unique viral nucleic acid sequences capable of inducing signaling and ER stress. This can lead to activate innate immune signaling via cGAS/STING and RIG1 pathways resulting in immune responses to viral and donor antigens resulting in CLAD.