Metabolism Of Fibroblasts Research Articles

Natural Exogenous Antioxidant Defense against Changes in Human Skin Fibroblast Proteome Disturbed by UVA Radiation.

Daily exposure of the skin to UVA radiation causes oxidative modifications to cellular components and biomolecules. These include proteins involved in the metabolism and cytoprotection of fibroblasts, and their modification can contribute to the disruption of cell function and the development of skin disorders. Therefore, there remains a need for highly active cytoprotective compounds with antioxidant properties. The purpose of this study was to investigate the effect of ascorbic acid on the activity of rutin against UVA-induced changes in the proteome of human fibroblasts. All analyses were carried out on fibroblasts cultured in a three-dimensional system exposed to UVA radiation and incubated with rutin and ascorbic acid. Their proteomic profile was analyzed using nano-HPLC, which revealed 150 proteins whose expression was significantly altered between treatment conditions. UVA radiation led to changes in the expression of 82 proteins. However, some of these changes were mitigated by rutin and ascorbic acid separately (23 and 25 proteins, respectively) and rutin and ascorbic acid together (23 proteins). UVA radiation has led to the upregulation of proteins involved in gene expression, catalytic processes and antioxidant pathways, and downregulation of proteins with binding activity. Nevertheless, rutin and ascorbic acid used separately or together have countered these changes to varying degrees. Moreover, rutin and ascorbic acid stimulated fibroblasts irradiated by UVA to increase the expression of the signalling molecules responsible for the opening of the transmembrane channels. In the context of the results obtained, the observed cytoprotective effect of the cooperation of rutin and ascorbic acid results not only from the overlapping properties of the compounds. The effect of rutin alone is probably inhibited by its limited bioavailability. Therefore, its interaction with ascorbic acid increases membrane penetration and improves the cytoprotective effect on skin fibroblasts.

Gingival fibroblasts dynamically reprogram cellular metabolism during infection of Porphyromonas gingivalis

ObjectiveThe purpose of the present study was to explore the sequential changes in the cellular metabolism in gingival fibroblasts (GFs) in response toPorphyromonas gingvalis (P. gingivalis) ATCC33277 infection. DesignGFs were treated withP. gingivalis at the MOI of 50 for 4, 24 and 48 h to mimic the early, medium, and late phase in the bacterial infection. LDH assay and cell counting kit-8 were utilized to explore cell death and proliferation. Real-time PCR was utilized to explore the gene transcription of pro-inflammatory genes. The relative levels of biomolecules in GFs were measured by gas chromatography-mass spectrometry. Principal component analysis and orthogonal partial least-squares-discriminant analysis were performed to visualize the metabolic difference among experimental groups. In addition, pathway analysis was conducted regarding differential metabolites in GFs. ResultsP. gingivalis infection triggered significant gene transcription of IL-1β, IL 6, MCP 1, and MMP 1 in GFs. In addition, P. gingivalis stimulated cell proliferation of GFs at MOI of 10, 50 and 250. Moreover, P. gingivalis triggered significant cell death at higher MOI. 69, 173 and 148 metabolites were qualitatively detected at 4, 24 and 48 h after P. gingivalis infection respectively in GFs, showing a sequential change of different phase. Kyoto Encyclopedia of Genes and Genomes pathway analysis demonstrated that ATP-binding cassette transporters, glutathione, purine and pyrimidine metabolism was significantly altered in different phase. ConclusionsHuman GFs may sequentially rewire metabolomics to shape the inflammatory responses and support the proliferation of host cells during P. gingivalis infection.

Comparative Redox-active Properties of Asparagine Metabolism in Zebrafish and Mouse Fibroblasts

Comparative Redox-active Properties of Asparagine Metabolism in Zebrafish and Mouse Fibroblasts

Leptin Receptor Signaling Regulates Protein Synthesis Pathways and Neuronal Differentiation in Pluripotent Stem Cells.

SummaryThe role of leptin receptor (OB-R) signaling in linking pluripotency with growth and development and the consequences of dysfunctional leptin signaling on progression of metabolic disease is poorly understood. Using a global unbiased proteomics approach we report that embryonic fibroblasts (MEFs) carrying the db/db mutation exhibit metabolic abnormalities, while their reprogrammed induced pluripotent stem cells (iPSCs) show altered expression of proteins involved in embryonic development. An upregulation in expression of eukaryotic translation initiation factor 4e (Eif4e) and Stat3 binding to the Eif4e promoter was supported by enhanced protein synthesis in mutant iPSCs. Directed differentiation of db/db iPSCs toward the neuronal lineage showed defects. Gene editing to correct the point mutation in db/db iPSCs using CRISPR-Cas9, restored expression of neuronal markers and protein synthesis while reversing the metabolic defects. These data imply a direct role for OB-R in regulating metabolism in embryonic fibroblasts and key developmental pathways in iPSCs.

TGF-β1 is a regulator of the pyruvate dehydrogenase complex in fibroblasts

TGF-β1 reprograms metabolism in renal fibroblasts, inducing a switch from oxidative phosphorylation to aerobic glycolysis. However, molecular events underpinning this are unknown. Here we identify that TGF-β1 downregulates acetyl-CoA biosynthesis via regulation of the pyruvate dehydrogenase complex (PDC). Flow cytometry showed that TGF-β1 reduced the PDC subunit PDH-E1α in fibroblasts derived from injured, but not normal kidneys. An increase in expression of PDH kinase 1 (PDK1), and reduction in the phosphatase PDP1, were commensurate with net phosphorylation and inactivation of PDC. Over-expression of mutant PDH-E1α, resistant to phosphorylation, ameliorated effects of TGF-β1, while inhibition of PDC activity with CPI-613 was sufficient to induce αSMA and pro-collagen I expression, markers of myofibroblast differentiation and fibroblast activation. The effect of TGF-β1 on PDC activity, acetyl-CoA, αSMA and pro-collagen I was also ameliorated by sodium dichloroacetate, a small molecule inhibitor of PDK. A reduction in acetyl-CoA, and therefore acetylation substrate, also resulted in a generalised loss of protein acetylation with TGF-β1. In conclusion, TGF-β1 in part regulates fibroblast activation via effects on PDC activity.

Dermal adipocytes contribute to the metabolic regulation of dermal fibroblasts.

Dermal fibroblasts are an essential population of skin cells. They are not only responsible for synthesis and remodelling of the extracellular matrix of the dermis, but also communicate with other skin cells via autocrine and paracrine interactions. Skin-associated dermal adipocytes reside below the reticular dermis. Strong lipolysis is observed during the regression of dermal adipocytes. However, the nature of the local intercellular crosstalk in which lipids released by dermal adipocytes affecting the metabolism of adjacent skin fibroblasts has not yet been examined. With the use of a series of novel mouse models that allow us to manipulate adipocytes, we demonstrate that dermal adipocytes can modulate the structure of the dermis through regulating extracellular matrix production in dermal fibroblasts. Fatty acids released by dermal adipocytes are involved in this process. Our observations offer new in vivo insights into the role of dermal adipocyte-derived lipids in influencing metabolism of adjacent local cells in the skin through a paracrine effect in the microenvironment of the dermal adipocyte.

An altered lipid metabolism characterizes Charcot-Marie-Tooth type 2B peripheral neuropathy

Charcot-Marie Tooth type 2B (CMT2B) is a rare inherited peripheral neuropathy caused by five missense mutations in the RAB7A gene, which encodes a small GTPase of the RAB family. Currently, no cure is available for this disease. In this study, we approached the disease by comparing the lipid metabolism of CMT2B-derived fibroblasts to that of healthy controls. We found that CMT2B cells showed increased monounsaturated fatty acid level and increased expression of key enzymes of monounsaturated and polyunsaturated fatty acid synthesis. Moreover, in CMT2B cells a higher expression of acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS), key enzymes of de novo fatty acid synthesis, with a concomitantly increased [1-14C]acetate incorporation into fatty acids, was observed. The expression of diacylglycerol acyltransferase 2, a rate-limiting enzyme in triacylglycerol synthesis, as well as triacylglycerol levels were increased in CMT2B compared to control cells. In addition, as RAB7A controls lipid droplet breakdown and lipid droplet dynamics have been linked to diseases, we analyzed these organelles and showed that in CMT2B cells there is a strong accumulation of lipid droplets compared to control cells, thus reinforcing our data on abnormal lipid metabolism in CMT2B. Furthermore, we demonstrated that ACC and FAS expression levels changed upon RAB7 silencing or overexpression in HeLa cells, thus suggesting that metabolic modifications observed in CMT2B-derived fibroblasts can be, at least in part, related to RAB7 mutations.

Effect of Regulating the Expression of HSP47 on Collagen Metabolism in Scleral Fibroblasts

ABSTRACT Purpose To explore the effect of heat shock protein 47 (HSP 47) on collagen (types I, III, and V) metabolism in scleral fibroblasts. Methods Scleral fibroblasts with over- or low-expression of HSP 47 were constructed by plasmid transfection. The mRNA and intracellular proteins expression of HSP 47, collagen (types I, III, and V) and α-smooth muscle actin (α-SMA) were detected by quantitative real-time polymerase chain reaction and western blot. The proteins expression of collagen (types I, III, and V), matrix metalloproteinase 2 (MMP-2), and tissue inhibitor of matrix metalloproteinases 1 and 2 (TIMP-1 and −2) in extracellular matrix (ECM) were detected by ELISA. The migration and proliferation activities of cells were detected by scratch-wound assay and MTS. The internal structure of scleral fibroblasts was observed by transmission electron microscopy (TEM). Results The results obtained demonstrated significant increases in the expression of the mRNA and protein expression of collagen I in HSP47 up-regulated cells. Overexpression of HSP 47 promotes the expression of α-SMA and cell migration. Down-regulated expression of HSP 47 results in decreased mRNA and protein expression of collagen. Low expression of HSP 47 significantly inhibits cell migration and proliferation, and affects the internal structure of cells. Conclusion HSP 47 affects collagen metabolism in scleral fibroblasts. It appears to promote the synthesis and secretion of collagen I as well as inhibit degradation.

Dissecting cell-type-specific metabolism in pancreatic ductal adenocarcinoma

Tumors are composed of many different cell types including cancer cells, fibroblasts, and immune cells. Dissecting functional metabolic differences between cell types within a mixed population can be challenging due to the rapid turnover of metabolites relative to the time needed to isolate cells. To overcome this challenge, we traced isotope-labeled nutrients into macromolecules that turn over more slowly than metabolites. This approach was used to assess differences between cancer cell and fibroblast metabolism in murine pancreatic cancer organoid-fibroblast co-cultures and tumors. Pancreatic cancer cells exhibited increased pyruvate carboxylation relative to fibroblasts, and this flux depended on both pyruvate carboxylase and malic enzyme 1 activity. Consequently, expression of both enzymes in cancer cells was necessary for organoid and tumor growth, demonstrating that dissecting the metabolism of specific cell populations within heterogeneous systems can identify dependencies that may not be evident from studying isolated cells in culture or bulk tissue.

Abstract B13: Utilizing metabolic reprogramming to regulate fibroblast phenotype and reduce radiation fibrosis

Abstract Introduction: Radiation fibrosis affects up to half of all patients undergoing radiotherapy for head and neck cancer treatment. It is characterized by excess extracellular matrix deposition, leading to tissue dysfunction, distortion, hardening, and pain. There are currently no effective treatments for radiation fibrosis. A fundamental challenge has been to understand and modulate the function of fibroblasts, a key mediator in extracellular matrix production and degradation. Recent evidence has demonstrated that metabolic regulation plays an important role in altering cell behavior in cancer biology and immunology. However, little is known about how metabolic changes affect fibroblast phenotype. Hypothesis: We propose that metabolic reprogramming may be an effective strategy to modulate fibroblast function and reduce radiation fibrosis. Results: An interplay between fatty acid oxidation and glycolysis was found to be a convergence point directly governing fibroblast behavior. It was demonstrated that manipulating the balance between FAO and glycolysis in fibroblasts induced either an extracellular matrix anabolic or catabolic phenotype. Specifically, anabolic fibroblasts relied on a fatty acid oxidation to glycolysis shift to produce extracellular matrix components. Reversal of this metabolic shift generated a catabolic fibroblast, which downregulated extracellular matrix synthesis and upregulated extracellular matrix lysosomal degradation. We further uncovered that CD36, a multifunctional fatty acid transporter, was a crucial link connecting fibroblast metabolism with extracellular matrix regulation, as its depletion completely inhibited collagen-1 internalization and degradation. Finally, through metabolic reprogramming using fibroblasts expressing high levels of CD36, but not CD36 knockout fibroblasts, metabolic balance could be restored, and in turn extracellular matrix accumulation was reduced in murine radiation fibrosis. Conclusions: We have uncovered that a fibroblast’s phenotype can be differentiated and regulated based on its metabolic signature. These findings have significant implications for drug development and for future cellular therapies to reduce radiation fibrosis. Citation Format: Xiao Zhao, Pamela Psarianos, David Goldstein, Ralph Gilbert, Ian Witterick, Laurie Ailles, Benjamin Haibe-Kains, Fei-Fei Liu. Utilizing metabolic reprogramming to regulate fibroblast phenotype and reduce radiation fibrosis [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr B13.

Deferiprone Stimulates Aged Dermal Fibroblasts via HIF-1α Modulation.

Hypoxia-inducible factor 1α (HIF-1α), a transcription factor responsible for tissue homeostasis and regeneration, presents reduced functionality in advanced age. In addition to absence of oxygen, sequestration of iron also stimulates HIF-1α. Therefore, we analyzed the efficacy of the iron-chelator deferiprone (DFP) at stimulating dermal fibroblasts. The main objective of this study was to quantify the DFP concentrations capable of stimulating dermal fibroblasts in vitro and to correlate the effective DFP concentrations with the ability of DFP to penetrate the epidermis, reach the dermis, and activate HIF-1α in vivo. We measured cell proliferation, metabolic activity, HIF-1α expression, and lactate dehydrogenase levels of both young and aged fibroblasts after a 24-hour in vitro preconditioning with DFP. In addition, we evaluated cell survival rates and morphology with different cellular stainings. Finally, we performed a transdermal permeation study with a 1% DFP topical formulation to quantify the concentration required to reach the dermis. In vitro administration of iron-chelation therapy (156-312.5 µg/mL DFP ) on aged fibroblasts resulted in activation of various antiaging processes. The concentration required to reach the dermis within 24 hours was 1.5% (0.15 mg/mL), which corresponds well with the effective doses of our laboratory analyses. The activation of HIF-1α by DFP enhances cell metabolism, proliferation, and survival of fibroblasts while reducing lactate dehydrogenase levels. Modulation of HIF-1α is linked to activation of key regeneration enzymes and proteins, and by proxy, antiaging. Therefore, the antiaging properties of DFP and its satisfactory dermal penetration make it a promising regenerative agent.

MTOR Signaling and SREBP Activity Increase FADS2 Expression and Can Activate Sapienate Biosynthesis

SummaryCancer cells display an increased plasticity in their lipid metabolism, which includes the conversion of palmitate to sapienate via the enzyme fatty acid desaturase 2 (FADS2). We find that FADS2 expression correlates with mammalian target of rapamycin (mTOR) signaling and sterol regulatory element-binding protein 1 (SREBP-1) activity across multiple cancer types and is prognostic in some cancer types. Accordingly, activating mTOR signaling by deleting tuberous sclerosis complex 2 (Tsc2) or overexpression of SREBP-1/2 is sufficient to increase FADS2 mRNA expression and sapienate metabolism in mouse embryonic fibroblasts (MEFs) and U87 glioblastoma cells, respectively. Conversely, inhibiting mTOR signaling decreases FADS2 expression and sapienate biosynthesis in MEFs with Tsc2 deletion, HUH7 hepatocellular carcinoma cells, and orthotopic HUH7 liver xenografts. In conclusion, we show that mTOR signaling and SREBP activity are sufficient to activate sapienate metabolism by increasing FADS2 expression. Consequently, targeting mTOR signaling can reduce sapienate metabolism in vivo.

Synthetic Hydroxyapatite Inhibits Bisphosphonate Toxicity to the Oral Mucosa In Vitro.

Medication-related osteonecrosis of the jaw (MRONJ) is a side effect of bisphosphonate therapy, characterised by exposed necrotic bone. The soft tissues of the oral mucosa no longer provide a protective barrier and MRONJ patients experience pain, infections and difficulties eating. We hypothesised that hydroxyapatite (Ca5(PO4)3(OH)) could reduce bisphosphonate concentrations and protect the oral mucosa by exploiting bisphosphonate’s calcium binding affinity. The effect of zoledronic acid (ZA) and pamidronic acid (PA) on the metabolism of oral fibroblasts, oral keratinocytes and three-dimensional oral mucosa models was investigated and then repeated in the presence of hydroxyapatite granules. Without hydroxyapatite, ZA and PA significantly reduced the metabolic activity of oral cells in a dose-dependent manner. Both drugs reduced epithelial thickness and 30 µM ZA resulted in loss of the epithelium. Hydroxyapatite granules had a protective effect on oral cells, with metabolic activity retained. Oral mucosa models retained their multi-layered epithelium when treated with ZA in the presence of hydroxyapatite granules and metabolic activity was comparable to controls. These results demonstrate hydroxyapatite granules protected oral soft tissues from damage caused by bisphosphonate exposure. Porous hydroxyapatite granules are currently used for socket preservation and this data suggests their potential to prevent MRONJ in at-risk patients.

Myotonic Dystrophy type 1 cells display impaired metabolism and mitochondrial dysfunction that are reversed by metformin.

Myotonic dystrophy type 1 (DM1; MIM #160900) is an autosomal dominant disorder, clinically characterized by progressive muscular weakness and multisystem degeneration. The broad phenotypes observed in patients with DM1 resemble the appearance of a multisystem accelerated aging process. However, the molecular mechanisms underlying these phenotypes remain largely unknown. In this study, we characterized the impact of metabolism and mitochondria on fibroblasts and peripheral blood mononuclear cells (PBMCs) derived from patients with DM1 and healthy individuals. Our results revealed a decrease in oxidative phosphorylation system (OXPHOS) activity, oxygen consumption rate (OCR), ATP production, energy metabolism, and mitochondrial dynamics in DM1 fibroblasts, as well as increased accumulation of reactive oxygen species (ROS). PBMCs of DM1 patients also displayed reduced mitochondrial dynamics and energy metabolism. Moreover, treatment with metformin reversed the metabolic and mitochondrial defects as well as additional accelerated aging phenotypes, such as impaired proliferation, in DM1-derived fibroblasts. Our results identify impaired cell metabolism and mitochondrial dysfunction as important drivers of DM1 pathophysiology and, therefore, reveal the efficacy of metformin treatment in a pre-clinical setting.

Study of the polyacrylate interpenetration in a collagen-polyurethane matrix to prepare novel hydrogels for biomedical applications

Currently, the control of the properties of collagen based hydrogels represents a promising area of research to develop novel materials for biomedical applications. The crosslinking of the collagen with trifunctional polyurethane (PU) allows a hybrid matrix to be formed by improving the coupling with exogenous polymeric chains to generate innovative semi-interpenetrated network (semi-IPN) hydrogels. The incorporation of polyacrylate (PA) within a hybrid matrix of collagen-PU allows to regulate the structure and physicochemical properties such as polymerization rate, physicochemical crosslinking, thermal stability, storage module and swelling/degradation behavior of the 3D matrices in the hydrogel state, also exhibiting modulation of their in vitro biocompatibility properties. This work contemplates the study of the effect of PA concentration on the physicochemical properties and the in vitro biological response of these novel semi-IPN hydrogels based on collagen-PU-PA. The results indicate that semi-IPN hydrogels that include 20 wt% of PA exhibit improved swelling with respect to the collagen-PU hydrogel, controlling the degradation rate in acidic, alkaline and proteolytic media; showing E. coli inhibition capacity, high hemocompatibility and not altering the metabolism of monocytes and fibroblasts growing on them. Therefore, these novel hydrogels represent biomaterials with potential application in biomedical strategies such as wound healing dressings.

Reprogramming of lipid metabolism in cancer-associated fibroblasts potentiates migration of colorectal cancer cells

Metabolic interaction between cancer-associated fibroblasts (CAFs) and colorectal cancer (CRC) cells plays a major role in CRC progression. However, little is known about lipid alternations in CAFs and how these metabolic reprogramming affect CRC cells metastasis. Here, we uncover CAFs conditioned medium (CM) promote the migration of CRC cells compared with normal fibroblasts CM. CAFs undergo a lipidomic reprogramming, and accumulate more fatty acids and phospholipids. CAFs CM after protein deprivation still increase the CRC cells migration, which suggests small molecular metabolites in CAFs CM are responsible for CRC cells migration. Then, we confirm that CRC cells take up the lipids metabolites that are secreted from CAFs. Fatty acids synthase (FASN), a crucial enzyme in fatty acids synthesis, is significantly increased in CAFs. CAF-induced CRC cell migration is abolished by knockdown of FASN by siRNA or reducing the uptake of fatty acids by CRC cells by sulfo-N-succinimidyloleate sodium in vitro and CD36 monoclonal antibody in vivo. To conclude, our results provide a new insight into the mechanism of CRC metastasis and suggest FASN of CAFs or CD36 of CRC cells may be potential targets for anti-metastasis treatment in the future.

Characterization of Collagen Metabolism in Normal and Chronic Obstructive Pulmonary Diseased Human Lung Fibroblasts Reared in Serum‐free Medium

The structural integrity of the human lung is maintained by a healthy extracellular matrix (ECM). One of the main components, collagen type I is produced by fibroblasts and provides strength and structure to the cells and the function of the lung. A careful balance of collagen degradation and production is controlled by matrix metalloproteases (MMPs). In diseased states such as Chronic Obstructive Pulmonary Disease (COPD), two collagenases, MMP‐8 and MMP‐12, are upregulated. In this study we measure collagen metabolism of human lung fibroblasts (HLFs) reared in serum‐free media (subconfluent normal (NHLF) and COPD diseased (DHLF)). Commercially available early passage human lung fibroblasts established from lung tissue, obtained during biopsy, were cultured and passaged in our laboratory through P4 and P5 for both the NHLF and DHLF. Both normal and diseased HLF cells were viable when cultured at subconfluence for 24 hours in serum free medium. They produced detectable quantities of intact collagen type I secreted and incorporated in the ECM. The amount of intact collagen decreased between passage 4 and 5 (p<0.01) in both cell types indicating there is a limit to the passages that can be utilized to measure ECM alterations. Degraded collagen type I was present in the cell layer fractions of both cell types, but was 10 fold higher in the cell layer of diseased HLF cells. This indicates that the expected ECM profile of diseased HLF can be demonstrated in serum‐free culture. Both cell types produced detectable levels of MMP‐8 and MMP‐12 collagenases. In this study we have demonstrated that normal and diseased HLF can be cultured in serum‐free conditions while maintaining their metabolic profile. The production of MMPs and collagens provides an experimental system in which we can test potential therapeutics that may alter the fibrotic response during COPD.Support or Funding InformationThis work is supported by a grant from the PCOM Alumni and the Division of Research at PCOM.

Mechanical strain affects collagen metabolism-related gene expression in scleral fibroblasts

We previously demonstrated that collagen metabolism affects scleral mechanical properties and scleral remodeling. Scleral remodeling changes the mechanical strain on sclera and scleral fibroblasts. We postulated that mechanical strain changes affect collagen metabolism in scleral fibroblasts. To understand the differences in collagen metabolism in scleral fibroblasts related to mechanical strain changes, scleral fibroblasts were isolated and cultured under different mechanical strains using the FX-4000 system or were treated with the TGF-β1 and TGFBR1 inhibitor LY364947. The collagen metabolism-related gene expression levels were detected. The results showed that the appropriate (lower) mechanical strain improved collagen synthesis and reduced collagen decomposition. In contrast, higher mechanical strain reduced collagen synthesis and enhanced collagen decomposition, especially a sustained higher strain. Furthermore, the effect of a transitory higher strain was recoverable, and collagen metabolism in scleral fibroblasts was regulated by TGF-β1. These results suggested that mechanical strain mediates TGF-β1 expression to regulate collagen metabolism in scleral fibroblasts, thereby affect scleral tissue remodeling.

Cartilage acidic protein 1 promotes increased cell viability, cell proliferation and energy metabolism in primary human dermal fibroblasts

Cartilage acidic protein 1 (CRTAC1) is an extracellular matrix protein of human chondrogenic tissue that is also present in other vertebrates, non-vertebrate eukaryotes and in some prokaryotes. The function of CRTAC1 remains unknown but the protein’s structure indicates a role in cell-cell or cell-matrix interactions and calcium-binding. The aim of the present study was to evaluate the in vitro effects of hCRTAC1-A on normal human dermal fibroblasts (NHDF). A battery of in vitro assays (biochemical and PCR), immunofluorescence and a biosensor approach were used to characterize the protein’s biological activities on NHDF cells in a scratch assay. Gene expression analysis revealed that hCRTAC1-A protein is associated with altered levels of expression for genes involved in the processes of cell proliferation (CXCL12 and NOS2), cell migration (AQP3 and TNC), and extracellular matrix-ECM regeneration and remodeling (FMOD, TIMP1, FN1) indicating a role for hCRTAC1-A in promoting these activities in a scratch assay. In parallel, the candidate processes identified by differential gene transcription were substantiated and extended using Electric cell-substrate impedance sensing (ECIS) technology, immunofluorescence and cell viability assays. Our findings indicate that hCRTAC1-A stimulated cell proliferation, migration and ECM production in primary human fibroblasts in vitro.

Genome-wide expression analysis of the heat stress response in dermal fibroblasts of Tharparkar (zebu) and Karan-Fries (zebu × taurine) cattle.

The present study sought to evaluate mRNA expression profiles in the cultured dermal fibroblasts of Tharparkar (zebu) and Karan-Fries (zebu, Tharparkar × taurine, Holstein Friesian) cattle in response to heat stress. Bioinformatics' analysis identified temperature-regulated biological processes and pathways. Biological processes overrepresented among the earliest genes induced by temperature stress include regulation of stress responses, protein repair, metabolism, protein transport, cell division, and apoptosis. The present microarray platform contains 51,338 synthesized oligonucleotide probes corresponding to at least 36,713 unigenes. A total of 11,183 and 8126 transcripts were differentially expressed with a fold change of ≥ 2 in Tharparkar and Karan-Fries cattle, respectively. Randomly selected real-time validation showed 83.33% correlation with microarray data. Functional annotation and pathway study of the differentially expressed transcripts or genes (DEGs) reveal that upregulated genes significantly (P < 0.05) affect protein processing and NOD-like receptor pathways (NLRs), while downregulated genes were significantly (P < 0.05) found to be associated with cell cycle, metabolism, and protein transport. Gene expression changes include activation of heat shock factors (HSFs), increased expression of heat shock proteins (HSPs), and apoptosis, while decreasing protein synthesis and another metabolism. These findings provide insights into the underlying mechanism of the physiology of heat stress in Tharparkar and Karan-Fries cattle. Understanding the biology and mechanisms of heat stress is critical to developing approaches to ameliorate current production issues for improving animal performance and agriculture economics in tropical climatic conditions. In conclusion, the present study indicates that heat stress differentially affects the expression of the significant number of genes associated with stress response, metabolism, apoptosis, and protein transport in dermal fibroblasts of Tharparkar and Karan-Fries cattle.