Protein Expression In Fibroblasts Research Articles

#1400 Acidosis-induced inflammatory response in tubule cells and fibroblasts is amplified by tubule-fibroblast crosstalk and mediated via p38 signaling

Abstract Background and Aims Tubulointerstitial kidney disease, associated with micromilieu changes such as acidification and inflammation, impacts tubule cells and fibroblasts. Micromilieu homeostasis influences intracellular signaling and intercellular crosstalk. Furthermore, cell-cell communication modulates the interstitial microenvironment. In the early stages of kidney failure, fibroblasts undergo a transition to an inflammatory phenotype driven by rapid and long-lasting p38 or CREB (cAMP response element-binding protein) signaling. This phenotypic shift involves an increased secretion of cytokines such as IL-6, TNF, or COX-2 metabolites, thereby changing the microenvironment. Additionally, the phenotype switch is characterized by remodelling of the cytoskeleton and alterations in cell-cell contacts. The objective of our study was to evaluate the impact of acidosis on inflammatory responses in proximal tubule cells and fibroblasts in the context of cellular crosstalk. Furthermore, we aimed to investigate the involvement of p38 and CREB signaling. Method HK-2 (human proximal tubule) and CCD-1092Sk (human fibroblasts), in mono and coculture, were exposed to acidic or control media for 3 or 48 h. Protein expression levels of inflammation markers (IL-6, TNF, TGF-ß, and COX-2), dedifferentiation markers (N-cadherin, vinculin, ß-catenin, and vimentin), as well as phospho- and total p38 and CREB, were determined through western blot analysis. The impact of p38 activation was assessed using pharmacological interventions. Furthermore, the influence of IL-6 incubation on the expression of phospho-p38 was investigated Results In tubule cells acidosis led, independent of culture conditions, to an increase of IL-6 after 3h and a decrease of vimentin and COX-2 after 48h. Moreover, acidosis caused an increase of TNF solely in coculture after 3h. Only in monoculture, ß-Catenin expression decreased in tubule cells during acidosis. In fibroblasts acidosis led to an increase of TNF, COX-2, vimentin, vinculin, N-cadherin and a decrease of TGF-ß expression exclusively in co-culture after 48 h. Independent of incubation or culture conditions acidosis induced a strong increase of IL-6 protein expression in fibroblasts. In co-culture, acidosis enhanced phosphorylation of p38 phosphorylation only after 3 h and that of CREB appeared after 3h and was persistent after 48h. In fibroblasts, acidosis enhanced phosphorylation of p38 in a sustained and very strong manner. Moreover, acidosis caused an increased phosphorylation of CREB independent of culture conditions and incubation time. Inhibition of p38 under co-culture conditions reduced acidosis-induced changes in fibroblasts significantly. In monoculture, incubation with IL-6 under acidic conditions caused an increase in p38 phosphorylation after 3 hours in tubule cells and after 48 hours in fibroblasts. Conclusion Our data indicate that acidosis-induced pathophysiological changes are significantly amplified by tubule-fibroblast crosstalk. The synergy between cellular crosstalk and acidosis triggers the activation of the p38, CREB, and IL-6 signaling pathways, leading to a phenotype switch in fibroblasts that promotes inflammation and fibrosis. If these findings hold true in vivo, the synergistic interplay between tubule-fibroblast crosstalk and acidosis emerges as a crucial factor in the development of kidney failure.

Natural Compounds Modulate Apolipoprotein E Gene and Protein Expression in Fibroblasts Derived from Young and Old Female Alzheimer’s Patients

Natural Compounds Modulate Apolipoprotein E Gene and Protein Expression in Fibroblasts Derived from Young and Old Female Alzheimer’s Patients

Evaluation of Novel Enhancer Compounds in Gentamicin-Mediated Readthrough of Nonsense Mutations in Rett Syndrome.

Rett syndrome (RTT), a severe X-linked neurodevelopmental disorder, is primarily caused by mutations in the methyl CpG binding protein 2 gene (MECP2). Over 35% RTT patients carry nonsense mutation in MECP2, making it a suitable candidate disease for nonsense suppression therapy. In our previous study, gentamicin was found to induce readthrough of MECP2 nonsense mutations with modest efficiency. Given the recent discovery of readthrough enhancers, CDX compounds, we herein evaluated the potentiation effect of CDX5-1, CDX5-288, and CDX6-180 on gentamicin-mediated readthrough efficiency in transfected HeLa cell lines bearing the four most common MECP2 nonsense mutations. We showed that all three CDX compounds potentiated gentamicin-mediated readthrough and increased full-length MeCP2 protein levels in cells expressing the R168X, R255X, R270X, and R294X nonsense mutations. Among all three CDX compounds, CDX5-288 was the most potent enhancer and enabled the use of reduced doses of gentamicin, thus mitigating the toxicity. Furthermore, we successfully demonstrated the upregulation of full-length Mecp2 protein expression in fibroblasts derived from Mecp2R255X/Y mice through combinatorial treatment. Taken together, findings demonstrate the feasibility of this combinatorial approach to nonsense suppression therapy for a subset of RTT patients.

Exosome Derived from Mesenchymal Stem Cells Alleviates Pathological Scars by Inhibiting the Proliferation, Migration and Protein Expression of Fibroblasts via Delivering miR-138-5p to Target SIRT1.

IntroductionThe therapies of using exosomes derived from mesenchymal stem cells (MSC-Exo) for wound healing and scar attenuation and micro RNAs (miRNAs) for regulation of genes by translational inhibition and mRNA destabilization obtained great achievements. Silent information regulator 1 (SIRT1) is the silent information, which has an intricate role in many biological processes. However, the effects of SIRT1 and miR-138-5p loaded in MSC-Exo on pathological scars remain unclear.MethodsMSC-Exo was isolated and identified by ultracentrifugation, transmission electron microscopy, nanoparticle size measuring instrument and Western blot assays. The relationship between SIRT1 and miR-138-5p was verified by a double-luciferase reporter assay. Cell Counting Kit-8, Τranswell, scratch, and Western blot assays were used to evaluate the proliferation and migration of human skin fibroblasts (HSFs), and the protein expression of SIRT1, NF-κB, α-SMA and TGF-β1 in HSFs, respectively. Flow cytometry was used to assess the apoptosis and cell cycle of HSFs affected by SIRT1.ResultsOur study demonstrated that miR-138-5p loaded in MSC-Exo could attenuate proliferation, migration and protein expression of HSFs-derived NF-κB, α-SMA, and TGF-β1 by targeting to SIRT1 gene, which confirmed the potential effects of MSC-Exo in alleviating pathological scars by performing as a miRNA’s delivery vehicle.ConclusionExosomes derived from MSCs acting as a delivery vehicle to deliver miR-138-5p can downregulate SIRT1 to inhibit the growth and protein expression of HSFs and attenuate pathological scars.

TAZ Reduces UVA-mediated Photoaging through Regulates Cell Proliferation in Skin Fibroblasts.

The transcriptional co-activator with PDZ-binding motif (TAZ) is a significant transcription factor downstream of the Hippo pathway regulating organ size, tissue regeneration, cell proliferation and apoptosis. Here, we report on TAZ in response to photoaging mediated by repeated UVA irradiation in skin fibroblasts. Continuous UVA irradiation caused a decrease in TAZ and targeted CTGF mRNA and protein expression in fibroblasts, accompanied by reduced cell proliferation, DNA damage, and cell cycle arrest in G1 phase and S phase reduction. Furthermore, P16 and P21 expression levels were increased, whereas Lamin B1 and Lamin A/C expression were decreased as a result of repeated UVA exposure. We further demonstrated that TAZ reduction enables photoaging caused by continuously UVA-irradiated fibroblasts. TAZ overexpression decreases G1 phase, augments the S phase and reduces P16 and P21 protein expression levels in fibroblasts. However, TAZ overexpressing cells exposed to chronic-UVA radiation show induced G1 phase arrest, an S phase reduction, and elevated P16 and P21 protein levels in fibroblasts, compared with TAZ overexpression cells. These findings suggest a novel function of TAZ to reduce photoaging in fibroblasts. This regulation implies that TAZ might be a viable therapeutic target for photoaging or UVA-related skin disorders.

TSLP-induced collagen type-I synthesis through STAT3 and PRMT1 is sensitive to calcitriol in human lung fibroblasts

Airway wall remodeling, a main pathology of asthma was linked to vitamin-D deficiency and protein arginine methyltransferase-1 (PRMT1) expression in sub-epithelial cell layers. Calcitriol reduced remodeling in asthma model, but its mode of action is unclear. This study assessed the effect of calcitriol on PRMT1-dependent fibroblast remodeling in human lung fibroblasts, and allergen-induced asthma in E3-rats. Fibroblasts were activated with thymic stromal lymphopoietin (TLSP); asthma was induced by ovalbumin inhalation in rats. The airway structure was assessed by immunohistology. Protein expression in fibroblasts and activation of the mitogen activated protein kinases were detected by Western-blotting. Transcription factor activation was determined by luciferase reporter assay. PRMT1 action was blocked by siRNA and PRMT-inhibition. Ovalbumin upregulated the expression of TSLP, PRMT1, matrix metallopro-teinase-1 (MMP1), interleukin-25, and collagen type-I in sub-epithelial fibroblasts. In isolated fibroblasts, TSLP induced the same proteins, which were blocked by inhibition of Erk1/2 and p38. TLSP induced PRMT1 through activation of signal transducer and activator of transcription-3. PRMT1 inhibition reduced collagen type-I expression and suppressed MMP1. In fibroblasts, calcitriol supplementation over 12 days prevented TSLP-induced remodeling by blocking the PRMT1 levels. Interestingly, short-term calcitriol treatment had no such effect. The data support the beneficial role of calcitriol in asthma therapy.

A unique esophageal extracellular matrix proteome alters normal fibroblast function in severe eosinophilic esophagitis

Eosinophilic esophagitis (EoE) is a chronic TH2 disorder complicated by tissue fibrosis and loss of esophageal luminal patency. The fibrostenotic esophagus does not respond well to therapy, but profibrotic therapeutic targets are largely unclear. Our aim was to utilize proteomics and primary cells as a novel approach to determine relevant profibrotic factors. We utilized primary esophageal EoE and normal fibroblasts, their derivative extracellular matrixes (ECMs), an approach of fibroblast culture on autologous versus nonautologous ECM, and proteomics to elucidate EoE ECM proteins that dysregulate cellular function. We cultured esophageal fibroblasts from normal esophagi and esophagi from patients with severe EoE on autologous versus nonautologous ECM. The EoE ECM proteome shifted normal esophageal fibroblast protein expression. Proteomic analysis demonstrated that thrombospondin-1 is detected only in the EoE ECM, is central in the EoE ECM protein-protein interactome, is found at significantly elevated levels in biopsy specimens from patients with active EoE, and induces fibroblast collagen I production. Fibroblasts from patients with EoE secrete a unique ECM proteome that reflects their invivo state and induces collagen I and α-smooth muscle actin protein expression from normal fibroblasts. Thrombospondin-1 isapreviously unappreciated profibrotic molecule in EoE.

Triptolide inhibits migration and proliferation of fibroblasts from ileocolonic anastomosis of patients with Crohn's disease via regulating the miR‑16‑1/HSP70 pathway.

Anastomotic fibrosis is highly likely to lead to reoperation in Crohn's disease (CD) patients. Triptolide (TPL) is considered to have anti-inflammatory and antifibrotic effects in a variety of autoimmune diseases, including CD. The present study aimed to investigate the effects of TPL on fibroblasts from strictured ileocolonic anastomosis of patients with CD and its underlying mechanism. Primary fibroblasts were obtained from strictured anastomosis tissue (SAT) samples and matched anastomosis-adjacent normal tissue (NT) samples which were collected from 10 CD patients who underwent reoperation because of anastomotic stricture. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to measure miR-16-1 and heat shock protein 70 (HSP70) levels. Western blotting was conducted to determine expression of HSP70, collagen I (Col-I), collagen III (Col-III) and α-smooth muscle actin (α-SMA) proteins. Agomir-16-1 and antagomir-16-1 were used to up and downregulate the expression of miR-16-1, respectively. Small interfering RNA (siRNA) was employed to inhibit the expression of HSP70. A wound healing assay was performed to measure the migration of fibroblasts. Cell proliferation was evaluated by MTT and 5-bromo-2-deoxyrudidine assays. Cell apoptosis was determined by caspase-3 activity and TUNEL assays. The results demonstrated that the levels of Col-I, Col-III and α-SMA were all significantly upregulated in SAT compared with NT. miR-16-1 levels in the SAT group were significantly compared with the NT group; conversely, the expression levels of HSP70 mRNA and protein in the SAT group were significantly lower compared with the NT group. Next, fibroblasts were treated with TPL to examine its effect on the miR-16-1/HSP70 pathway. The results demonstrated that the elevated expression of miR-16-1 in the SAT group was effectively inhibited by TPL treatment. Compared with the NT group, both the mRNA and protein levels of HSP70 were significantly downregulated in the SAT group cells, while TPL exhibited a strong promoting effect on HSP70 synthesis. Furthermore, upregulation of miR-16-1 reversed the effect of TPL on the miR-16-1/HSP70 pathway in fibroblasts from SAT. Overexpression of miR-16-1 significantly reversed the inhibitory effects of TPL treatment on migration, proliferation and extracellular matrix (ECM)-associated protein expression of fibroblasts from SAT. Finally, downregulation of miR-16-1 caused similar effects to the fibroblasts as the TPL treatment; however, the inhibitory effects on cell biological functions induced by antagomir-16-1 were all significantly reversed by HSP70 silencing. The present findings indicated that TPL may be a potential therapeutic option for postoperative anastomosis fibrosis of patients with CD. The miR-16-1/HSP70 pathway had a substantial role in the inhibitory effects of TPL on migration, proliferation and ECM synthesis rate of fibroblasts from strictured anastomosis tissues.

Effect of Pressure Stresses on Cell Viability and Protein Expression of Fascial Fibroblast.

BackgroundMany physical and mechanical phenomena occur during the acupuncture and tuina regime, and pressure is one of the most basic mechanical phenomena.ObjectivesTo understand the cellular bio-physical mechanism of basic mechanical stimulation via acupuncture and tuina by investigating the effect of different in vitro pressures on the cell viability and protein expression differences that originate from the facial fibroblasts around the meridians.Materials and MethodsIn vitro culture of the facial fibroblasts around the meridians was conducted using different pressures to perform single and multiple stimulation(s) on the cells. Thus, the changes in the fibroblast cell viability (cell viability rate and diameter) were tested, and changes in the fibroblast protein expression were observed.ResultsWe found that the pressure stimulation may excite the fascial fibroblast viability at the acupoint and increase cell viability. Two interactive factors are involved: the pressure intensity and the number of pressure stimulations. In addition, we found that all three pressures lead to significant regulation effects on the protein expression of the meridian-related fascial tissue fibroblasts, and clustering analysis revealed that 100 kPa pressure stimulation exhibits the most evident effect on the protein expression which is the pressure inducing the most differentiated protein expression.ConclusionsDuring the in vitro pressure process, the difference in the cell viability rate and protein expression of the facial fibroblasts around the meridians may (from a cell mechanics’ point-of-view) reveal the cytobiological and therapeutic mechanism of the basic mechanical stimulation via acupuncture and tuina on the facial fibroblasts around the meridians.

Polydatin attenuates reactive oxygen species-induced airway remodeling by promoting Nrf2-mediated antioxidant signaling in asthma mouse model

Reactive oxygen species (ROS) and epithelial–mesenchymal transition (EMT) play a critical role in transforming growth factor (TGF)-β1-mediated fibrotic airway remodeling in asthma. Polydatin (PD) is a small natural molecule in Chinese medicine; it is isolated from Polygonum cuspidatum and has antioxidative properties. In this study, we aimed to determine whether PD was protective against ROS-induced pulmonary fibrosis in asthma. Ovalbumin (OVA) was used to induce asthma in a mouse model that was treated with or without PD. We also created nuclear factor erythroid 2-related factor 2 (Nrf2) knockdown BEAS-2B cells and investigated whether PD reversed TGF-β1-induced pulmonary epithelial cell EMT by promotion of Nrf2-mediated antioxidation. Immunofluorescence showed that ROS and TGF-β1 expression was significantly increased in lung tissue from the OVA-induced asthma model. PD treatment inhibited activity of ROS and TGF-β1. Immunohistochemistry showed that PD treatment decreased OVA-induced lung ROS, TGF-β1 expression and fibroblasts. Western blotting showed that PD treatment reversed OVA-induced NADPH oxidase (NOX)1/4 expression by promoting Nrf2-mediated heme oxygenase-1 and NADPH dehydrogenase (quinone)-1 expression. PD treatment suppressed OVA-induced EMT and lung fibroblast protein expression in lung tissue. Nrf2 downregulation suppressed the protective effect of PD by promoting TGF-β1-induced ROS and EMT and accumulation of extracellular-matrix-related protein. All these data indicate that PD has potential therapeutic effects in asthma by promoting Nrf2-mediated antioxidation.

Loss of tenascin X gene function impairs injury-induced stromal angiogenesis in mouse corneas.

To determine the contribution by tenascin X (Tnx) gene expression to corneal stromal angiogenesis, the effects were determined of its loss on this response in TNX knockout (KO) mice. In parallel, the effects of such a loss were evaluated on vascular endothelial growth factor (VEGF) and transforming growth factor β1 (TGFβ1) gene and protein expression in fibroblasts and macrophages in cell culture. Histological, immunohistochemical and quantitative RT‐PCR changes determined if Tnx gene ablation on angiogenic gene expression, inflammatory cell infiltration and neovascularization induced by central corneal stromal cauterization. The role was determined of Tnx function in controlling VEGF‐A or TGFβ1 gene expression by comparing their expression levels in ocular fibroblasts and macrophages obtained from wild‐type (WT) and body‐wide Tnx KO mice. Tnx was up‐regulated in cauterized cornea. In Tnx KO, macrophage invasion was attenuated, VEGF‐A and its cognate receptor mRNA expression along with neovascularization were lessened in Tnx KOs relative to the changes occurring in their WT counterpart. Loss of Tnx instead up‐regulated in vivo mRNA expression of anti‐angiogenic VEGF‐B but not VEGF‐A. On the other hand, TGFβ1 mRNA expression declined in Tnx KO cultured ocular fibroblasts. Loss of Tnx gene expression caused VEGF‐A expression to decline in macrophages. Tnx gene expression contributes to promoting TGFβ1 mRNA expression in ocular fibroblasts and VEGF‐A in macrophages, macrophage invasion, up‐regulation of VEGF‐A expression and neovascularization in an injured corneal stroma. On the other hand, it suppresses anti‐angiogenic VEGF‐B mRNA expression in vivo.

Strain Stimulations with Different Intensities on Fibroblast Viability and Protein Expression

AbstractBackgroundMechanical stimulation via acupuncture and tuina massage triggers various cell responses. This study aims to understand these cellular bio-physical mechanisms by investigating the effect of different stimulation intensities on cell viability and protein expression.MethodologyConnective tissue fibroblasts were cultured in vitro. Three varying intensities of mechanical strain stimulation were applied to the cells, either once or three times and compared with non-stimulated controls. Changes in fibroblast viability and fibroblast protein expression were observed.ResultsStrain stimulation intensity significantly increased fibroblast cell survival rate (p<0.01) to effectively improve cell viability. Moreover, the combined influence of both the strain stimulation intensity and number of stimulations on the fibroblast survival rate significantly differed (p<0.05). Strain intensity also significantly altered fibroblast protein expression between the three groups (p<0.0001). Cluster analysis showed that the medium-intensity strain stimulation posed the maximum influence on protein expression.ConclusionThe difference in cell viability and protein expression of the connective tissue fibroblast during the in vitro strain process reveals the cytobiological mechanism of basic medicinal mechanical stimulation.

Aging decreases collagen IV expression in vivo in the dermo-epidermal junction and in vitro in dermal fibroblasts: possible involvement of TGF-β1.

Collagen IV is a major component of the dermo-epidermal junction (DEJ). To study expression of collagen IV upon aging in the DEJ and dermal fibroblasts isolated from the same patients. A model of senescent fibroblasts was developed in order to identify biological compounds that might restore the level of collagen IV. Skin fragments of women (30 to 70 years old) were collected. Localisation of collagen IV expression in the DEJ was studied by immunofluorescence. Fibroblast collagen IV expression was studied by real-time PCR, ELISA, and western blotting. Premature senescence was simulated by exposing fibroblasts to subcytotoxic H2O2 concentrations. Collagen IV decreased in the DEJ and fibroblasts relative to age. TGF-β1 treatment significantly increased collagen IV gene and protein expression in fibroblasts and restored expression in the model of senescence. Addition of TGF-β1-neutralizing antibody to fibroblast cultures decreased collagen IV expression. Taken together, the results suggest that the decrease in collagen IV in the DEJ, relative to age, could be due to a decrease in collagen IV expression by senescent dermal fibroblasts and may involve TGF-β1 signalling.

Fibroblast viability and phenotypic changes within glycated stiffened three-dimensional collagen matrices.

BackgroundThere is growing interest in the development of cell culture assays that enable the rigidity of the extracellular matrix to be increased. A promising approach is based on three-dimensional collagen type I matrices that are stiffened by cross-linking through non-enzymatic glycation with reducing sugars.MethodsThe present study evaluated the biomechanical changes in the non-enzymatically glycated type I collagen matrices, including collagen organization, the advanced glycation end products formation and stiffness achievement. Gels were glycated with ribose at different concentrations (0, 5, 15, 30 and 240 mM). The viability and the phenotypic changes of primary human lung fibroblasts cultured within the non-enzymatically glycated gels were also evaluated along three consecutive weeks. Statistical tests used for data analyze were Mann–Whitney U, Kruskal Wallis, Student’s t-test, two-way ANOVA, multivariate ANOVA, linear regression test and mixed linear model.ResultsOur findings indicated that the process of collagen glycation increases the stiffness of the matrices and generates advanced glycation end products in a ribose concentration-dependent manner. Furthermore, we identified optimal ribose concentrations and media conditions for cell viability and growth within the glycated matrices. The microenvironment of this collagen based three-dimensional culture induces α-smooth muscle actin and tenascin-C fibroblast protein expression. Finally, a progressive contractile phenotype cell differentiation was associated with the contraction of these gels.ConclusionsThe use of non-enzymatic glycation with a low ribose concentration may provide a suitable model with a mechanic and oxidative modified environment with cells embedded in it, which allowed cell proliferation and induced fibroblast phenotypic changes. Such culture model could be appropriate for investigations of the behavior and phenotypic changes in cells that occur during lung fibrosis as well as for testing different antifibrotic therapies in vitro.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-015-0237-z) contains supplementary material, which is available to authorized users.

Dysferlinopathy Fibroblasts Are Defective in Plasma Membrane Repair.

Background: Dysferlin is a sarcolemmal protein that is defective in Miyoshi myopathy and limb-girdle muscular dystrophy type 2B, and is involved in sarcolemmal repair. Primary cultured myoblasts and myotubes established from patient muscle biopsies have been widely utilized to explore the molecular mechanism of dysferlinopathy.Objectives: The purpose of this study was to explore the possible utility of dermal fibroblasts from dysferlin-deficient patients and SJL mice as a tool for studying dysferlinopathy.Methods: Dysferlin protein expression in fibroblasts from dysferlin-deficient patients and SJL mice was analyzed by immunoblotting and immunocytochemistry. The membrane wound-repair assay was performed on the fibroblasts using a confocal microscope equipped with a UV-laser. The membrane blebbing assay using hypotonic shock, in which normal membrane blebbing is detected only in the presence of dysferlin, was also performed using human and mouse fibroblasts.Results: Mis-sense mutated dysferlin was expressed at a very low level in fibroblasts from a dysferlinopathy patient, and lower expression level of truncated dysferlin was observed in SJL mouse fibroblast. Fibroblasts from patients with dysferlinopathy and SJL mice showed attenuated membrane repair and did not form membrane blebs in response to hypoosmotic shock. Proteosomal inhibitior increased mis-sense mutated or truncated dysferlin levels, and restored membrane blebbing, however, proteosomal inhibition failed to improve levels of dysferlin with non-sense or frame-shift mutation.Conclusion: Fibroblasts from dysferlinopathy patients and SJL mice showed attenuated plasma membrane repair, and could be a tool for studying dysferlinopathy.

A novel β-adrenergic response element regulates both basal and agonist-induced expression of cyclin-dependent kinase 1 gene in cardiac fibroblasts

Cardiac fibrosis, a known risk factor for heart disease, is typically caused by uncontrolled proliferation of fibroblasts and excessive deposition of extracellular matrix proteins in the myocardium. Cyclin-dependent kinase 1 (CDK1) is involved in the control of G2/M transit phase of the cell cycle. Here, we showed that isoproterenol (ISO)-induced cardiac fibrosis is associated with increased levels of CDK1 exclusively in fibroblasts in the adult mouse heart. Treatment of primary embryonic ventricular cell cultures with ISO (a nonselective β-adrenergic receptor agonist) increased CDK1 protein expression in fibroblasts and promoted their cell cycle activity. Quantitative PCR analysis confirmed that ISO increases CDK1 transcription in a transient manner. Further, the ISO-responsive element was mapped to the proximal -100-bp sequence of the CDK1 promoter region using various 5'-flanking sequence deletion constructs. Sequence analysis of the -100-bp CDK1 minimal promoter region revealed two putative nuclear factor-Y (NF-Y) binding elements. Overexpression of the NF-YA subunit in primary ventricular cultures significantly increased the basal activation of the -100-bp CDK1 promoter construct but not the ISO-induced transcription of the minimal promoter construct. In contrast, dominant negative NF-YA expression decreased the basal activity of the minimal promoter construct and ISO treatment fully rescued the dominant negative effects. Furthermore, site-directed mutagenesis of the distal NF-Y binding site in the -100-bp CDK1 promoter region completely abolished both basal and ISO-induced promoter activation of the CDK1 gene. Collectively, our results raise an exciting possibility that targeting CDK1 or NF-Y in the diseased heart may inhibit fibrosis and subsequently confer cardioprotection.

Abstract P3-01-06: TIGAR induces two compartment metabolic coupling and tamoxifen resistance

Abstract We have previously demonstrated that metabolic coupling occurs in a subgroup of human breast cancers between fibroblasts and carcinoma cells. In metabolically coupled tumors, cancer associated fibroblasts (CAFs) have low Cav-1 protein expression, are highly glycolytic and generate large amounts of lactate which is then released to the extracellular space via increased expression of monocarboxylate transporter 4 (MCT4) compared to normal fibroblasts (NFs). Conversely, in metabolically coupled tumors, carcinoma cells have low glycolysis with high uptake of lactate via monocarboxylate transporter 1 (MCT1) and high mitochondrial oxidative phosphorylation (OXPHOS) using lactate as a substrate compared to non-carcinoma epithelial cells. We have identified two fibroblast markers of metabolically coupled breast cancer: low caveolin-1 (Cav-1) protein expression and high MCT4 protein expression. Low Cav-1 expression and high MCT4 expression in breast cancer fibroblasts are prognostic biomarkers associated with poor outcomes. However, the mechanisms by which carcinoma cells reprogram CAFs to induce loss of fibroblast Cav-1 or increased expression of MCT4 protein are unknown. TP53 inducible glycolysis and apoptosis regulator (TIGAR) is a p53 regulated protein that inhibits glycolysis and induces OXPHOS. We investigated the role of TIGAR on expression of the fibroblast prognostic biomarkers Cav-1 and MCT4. We immunohistochemically stained 10 human ER+ breast cancer samples for TIGAR. TIGAR protein expression was highest in the carcinoma cells compared to fibroblasts and non-carcinoma epithelial cells in all 10 samples. We then cocultured T47D ER+ breast cancer cells with human fibroblasts (BJ-1) and discovered that the presence of fibroblasts was sufficient to increase TIGAR protein expression in carcinoma cells (1.5 fold, p<0.05). This coculture system was also sufficient to downregulate Cav-1 protein expression in fibroblasts (2.3 fold, p<0.01). We generated fibroblasts with Cav-1 shRNA knockdown and discovered that TIGAR protein expression decreased. We generated TIGAR overexpressing T47D cells and found that they had higher OXPHOS activity. Coculture of TIGAR overexpressing T47D cells with fibroblasts induced further downregulation of Cav-1 protein in fibroblasts (3.2 fold, p<0.05) and increased MCT4 protein in fibroblasts (2 fold, p<0.05) compared to coculture with control T47D cells. Overexpression of TIGAR in T47D cells was sufficient to increase 2-deoxyglucose uptake in fibroblasts by 1.2 fold (p<0.01) and decrease 2-deoxyglucose uptake in T47D cells by 1.3 fold (p<0.05). 2-deoxyglycose uptake is a marker of glycolysis. Finally, resistance to baseline apoptosis (1.6 fold,p<0.001) and with tamoxifen (1.2 fold, p<0.01) was induced by TIGAR overexpression. In summary, TIGAR expression in carcinoma cells is sufficient to induce carcinoma cell resistance to apoptosis, decrease Cav-1 and increase MCT4 protein expression in fibroblasts. TIGAR should be studied as a prognostic and predictive biomarker in breast cancer and a potential drug target. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-01-06.

389. TALEFrat#8–VP64 Increases Frataxin Gene and Protein Expression in Fibroblasts of Friedreich Patients and of Mouse Model

389. TALEFrat#8–VP64 Increases Frataxin Gene and Protein Expression in Fibroblasts of Friedreich Patients and of Mouse Model

Effects of combined radiation and burn injury on the renin–angiotensin system

The renin-angiotensin system (RAS) plays an important role in wound repair; however, little is known pertaining to RAS expression in response to thermal injury and the combination of radiation plus burn injury (CRBI). The purpose of this study was to test the hypothesis that thermal injury modifies expression of RAS components and CRBI delayed this up-regulation of RAS. Skin from uninjured mice was compared with mice receiving local thermal injury or CRBI (injury site). Skin was analyzed for gene and protein expression of RAS components. There was an initial increase in the expression of various components of RAS following thermal injury. However, in the higher CRBI group there is an initial decrease in AT(1b) (vasoconstriction, pro-proliferative), AT(2) (vasodilation, differentiation), and Mas (vasodilation, anti-inflammatory) gene expression. This corresponded with a delay and decrease in AT(1) , AT(2) , and MAS protein expression in fibroblasts and keratinocytes. The reduction in RAS receptor positive fibroblasts and keratinocytes correlated with a reduction in collagen deposition and keratinocyte infiltration into the wounded area resulting in a delay of reepithelialization following CRBI. These data support the hypothesis that delayed wound healing observed in subjects following radiation exposure may be in part due to decreased expression of RAS.

BRCA1 mutations drive oxidative stress and glycolysis in the tumor microenvironment

Mutations in the BRCA1 tumor suppressor gene are commonly found in hereditary breast cancer. Similarly, downregulation of BRCA1 protein expression is observed in the majority of basal-like breast cancers. Here, we set out to study the effects of BRCA1 mutations on oxidative stress in the tumor microenvironment. To mimic the breast tumor microenvironment, we utilized an in vitro co-culture model of human BRCA1-mutated HCC1937 breast cancer cells and hTERT-immortalized human fibroblasts. Notably, HCC1937 cells induce the generation of hydrogen peroxide in the fibroblast compartment during co-culture, which can be inhibited by genetic complementation with the wild-type BRCA1 gene. Importantly, treatment with powerful antioxidants, such as NAC and Tempol, induces apoptosis in HCC1937 cells, suggesting that microenvironmental oxidative stress supports cancer cell survival. In addition, Tempol treatment increases the apoptotic rates of MDA-MB-231 cells, which have wild-type BRCA1, but share a basal-like breast cancer phenotype with HCC1937 cells. MCT4 is the main exporter of L-lactate out of cells and is a marker for oxidative stress and glycolytic metabolism. Co-culture with HCC1937 cells dramatically induces MCT4 protein expression in fibroblasts, and this can be prevented by either BRCA1 overexpression or by pharmacological treatment with NAC. We next evaluated caveolin-1 (Cav-1) expression in stromal fibroblasts. Loss of Cav-1 is a marker of the cancer-associated fibroblast (CAF) phenotype, which is linked to high stromal glycolysis, and is associated with a poor prognosis in numerous types of human cancers, including breast cancers. Remarkably, HCC1937 cells induce a loss of Cav-1 in adjacent stromal cells during co-culture. Conversely, Cav-1 expression in fibroblasts can be rescued by administration of NAC or by overexpression of BRCA1 in HCC1937 cells. Notably, BRCA1-deficient human breast cancer samples (9 out of 10) also showed a glycolytic stromal phenotype, with intense mitochondrial staining specifically in BRCA1-deficient breast cancer cells. In summary, loss of BRCA1 function leads to hydrogen peroxide generation in both epithelial breast cancer cells and neighboring stromal fibroblasts, and promotes the onset of a reactive glycolytic stroma, with increased MCT4 and decreased Cav-1 expression. Importantly, these metabolic changes can be reversed by antioxidants, which potently induce cancer cell death. Thus, antioxidant therapy appears to be synthetically lethal with a BRCA1-deficiency in breast cancer cells and should be considered for future cancer prevention trials. In this regard, immunostaining with Cav-1 and MCT4 could be used as cost-effective biomarkers to monitor the response to antioxidant therapy.