Receptor regulation of phosphoinositide 3-hydroxykinase in the NG115-401L-C3 neuronal cell line: stimulation by insulin-like growth factor-I

The activation of phosphoinositide 3-hydroxykinase (P13K) is currently believed to represent the critical regulatory event which leads to the production of a novel intracellular signal. We have examined the control of this pathway by a number of cell-surface receptors in NG115-401L-C3 neuronal cells. Insulin-like growth factor-I stimulated the accumulation of 3-phosphorylated inositol lipids in intact cells and the appearance of P13K in antiphosphotyrosine-antibody-directed immunoprecipitates prepared from lysed cells, suggesting that P13K had been activated by a mechanism involving a protein tyrosine kinase. In contrast, P13K in these cells was not regulated by a variety of G-protein-coupled receptors, nerve growth factor acting via a low affinity receptor, or receptors for transforming growth factor-beta and interleukin-1. The receptor-specificity of P13K activation in these cells places significant constraints on the possible physiological function(s) of this pathway.

Role of the dsRNA-dependent protein kinase (PKR) in the attenuation of protein loss from muscle by insulin and insulin-like growth factor-I (IGF-I)

Proteolysis-inducing factor (PIF), a tumour-produced cachectic factor, induced a dose-dependent decrease in protein synthesis in murine myotubes, together with an increase in phosphorylation of eucaryotic initiation factor 2 (eIF2) on the alpha-subunit. Both insulin (1 nM) and insulin-like growth factor I (IGF-I) (13.2 nM) attenuated the depression of protein synthesis by PIF and the increased phosphorylation of eIF2alpha, by inhibiting the activation (autophosphorylation) of the dsRNA-dependent protein kinase (PKR) by induction of protein phosphatase 1. A low-molecular weight inhibitor of PKR also reversed the depression of protein synthesis by PIF to the same extent, as did insulin and IGF-I. Both insulin and IGF-I-stimulated protein synthesis in the presence of PIF, and this was attenuated by Salubrinal, an inhibitor of phospho eIF2alpha phosphatase, suggesting that at least part of this action was due to their ability to inhibit phosphorylation of eIF2alpha. Both insulin and IGF-I also attenuated the induction of protein degradation in myotubes induced by PIF, this effect was also attenuated by Salubrinal. These results suggest an alternative mechanism involving PKR to explain the effect of insulin and IGF-I on protein synthesis and degradation in skeletal muscle in the presence of catabolic factors.

MicroRNA-155 promotes resolution of hypoxia-inducible factor 1α activity during prolonged hypoxia

The hypoxia-inducible factor (HIF) is a key regulator of the transcriptional response to hypoxia. While the mechanism underpinning HIF activation is well understood, little is known about its resolution. Both the protein and the mRNA levels of HIF-1a (but not HIF-2a) were decreased in intestinal epithelial cells exposed to prolonged hypoxia. Coincident with this, microRNA (miRNA) array analysis revealed multiple hypoxiainducible miRNAs. Among these was miRNA-155 (miR-155), which is predicted to target HIF-1a mRNA. We confirmed the hypoxic upregulation of miR-155 in cultured cells and intestinal tissue from mice exposed to hypoxia. Furthermore, a role for HIF-1a in the induction of miR-155 in hypoxia was suggested by the identification of hypoxia response elements in the miR-155 promoter and confirmed experimentally. Application of miR-155 decreased the HIF-1a mRNA, protein, and transcriptional activity in hypoxia, and neutralization of endogenous miR-155 reversed the resolution of HIF-1a stabilization and activity. Based on these data and a mathematical model of HIF-1a suppression by miR-155, we propose that miR-155 induction contributes to an isoform-specific negative-feedback loop for the resolution of HIF-1a activity in cells exposed to prolonged hypoxia, leading to oscillatory behavior of HIF-1a-dependent transcription. © 2011, American Society for Microbiology.

A dynamic model of the hypoxia-inducible factor 1α (HIF-1α) network

Activation of the hypoxia-inducible factor (HIF) pathway is a critical step in the transcriptional response to hypoxia. Although many of the key proteins involved have been characterised, the dynamics of their interactions in generating this response remain unclear. In the present study, we have generated a comprehensive mathematical model of the HIF-1a pathway based on core validated components and dynamic experimental data, and confirm the previously described connections within the predicted network topology. Our model confirms previous work demonstrating that the steps leading to optimal HIF-1a transcriptional activity require sequential inhibition of both prolyl- and asparaginyl-hydroxylases. We predict from our model (and confirm experimentally) that there is residual activity of the asparaginyl-hydroxylase FIH (factor inhibiting HIF) at low oxygen tension. Furthermore, silencing FIH under conditions where prolyl-hydroxylases are inhibited results in increased HIF-1a transcriptional activity, but paradoxically decreases HIF-1a stability. Using a core module of the HIF network and mathematical proof supported by experimental data, we propose that asparaginyl hydroxylation confers a degree of resistance upon HIF-1a to proteosomal degradation. Thus, through in vitro experimental data and in silico predictions, we provide a comprehensive model of the dynamic regulation of HIF-1a transcriptional activity by hydroxylases and use its predictive and adaptive properties to explain counter-intuitive biological observations.

Hypoxia-inducible factor (HIF) network:insights from mathematical models

Oxygen is a crucial molecule for cellular function. When oxygen demand exceeds supply, the oxygen sensing pathway centred on the hypoxia inducible factor (HIF) is switched on and promotes adaptation to hypoxia by up-regulating genes involved in angiogenesis, erythropoiesis and glycolysis. The regulation of HIF is tightly modulated through intricate regulatory mechanisms. Notably, its protein stability is controlled by the oxygen sensing prolyl hydroxylase domain (PHD) enzymes and its transcriptional activity is controlled by the asparaginyl hydroxylase FIH (factor inhibiting HIF-1).To probe the complexity of hypoxia-induced HIF signalling, efforts in mathematical modelling of the pathway have been underway for around a decade. In this paper, we review the existing mathematical models developed to describe and explain specific behaviours of the HIF pathway and how they have contributed new insights into our understanding of the network. Topics for modelling included the switch-like response to decreased oxygen gradient, the role of micro environmental factors, the regulation by FIH and the temporal dynamics of the HIF response. We will also discuss the technical aspects, extent and limitations of these models. Recently, HIF pathway has been implicated in other disease contexts such as hypoxic inflammation and cancer through crosstalking with pathways like NF?B and mTOR. We will examine how future mathematical modelling and simulation of interlinked networks can aid in understanding HIF behaviour in complex pathophysiological situations. Ultimately this would allow the identification of new pharmacological targets in different disease settings.

Mechanism of attenuation of angiotensin-II-induced protein degradation by insulin-like growth factor-I (IGF-I)

Insulin-like growth factor-I (IGF-I) has been shown to attenuate protein degradation in murine myotubes induced by angiotensin II through downregulation of the ubiquitin-proteasome pathway, although the mechanism is not known. Angiotensin II is known to upregulate this pathway through a cellular signalling mechanism involving release of arachidonic acid, activation of protein kinase Cα (PKCα), degradation of inhibitor-κB (I-κB) and nuclear migration of nuclear factor-κB (NF-κB), and all of these events were attenuated by IGF-I (13.2 nM). Induction of the ubiquitin-proteasome pathway has been linked to activation of the RNA-activated protein kinase (PKR), since an inhibitor of PKR attenuated proteasome expression and activity in response to angiotensin II and prevented the decrease in the myofibrillar protein myosin. Angiotensin II induced phosphorylation of PKR and of the eukaryotic initiation factor-2 (eIF2) on the α-subunit, and this was attenuated by IGF-I, by induction of the expression of protein phosphatase 1, which dephosphorylates PKR. Release of arachidonic acid and activation of PKCα by angiotensin II were attenuated by an inhibitor of PKR and IGF-I, and the effect was reversed by Salubrinal (15 μM), an inhibitor of eIF2α dephosphorylation, as was activation of PKCα. In addition myotubes transfected with a dominant-negative PKR (PKRΔ6) showed no release of arachidonate in response to Ang II, and no activation of PKCα. These results suggest that phosphorylation of PKR by angiotensin II was responsible for the activation of the PLA2/PKC pathway leading to activation of NF-κB and that IGF-I attenuates protein degradation due to an inhibitory effect on activation of PKR. © 2007 Elsevier Inc. All rights reserved.

Insulin-like growth factor-I gene expression as a growth indicator in Nile tilapia Oreochromis niloticus L.

Somatic growth in fishes is regulated by a variety of hormones. A central step in this hormonal network is the growth hormone-insulin-like growth factor-I (IGF-I) axis. Studies conducted evaluated the relationship of hepatic IGF-I (hIGF-1) mRNA with growth as affected by feeding regimes (satiation or restricted level; daily or alternate-day feeding), temperatures (high, ambient, low) and by social stress. To develop a cellular means for the quantification of hIGF-I mRNA levels in O. niloticus, hIGF-I cDNA was isolated and cloned. The partial sequence of IGF-I cDNA encodes for signal peptide, mature protein and a portion of the E-domain. A sensitive TaqMan quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed based on the mature IGF-I. Using the developed qRT-PCR assay a significant positive correlation was observed between hIGF-I mRNA levels and growth rate of fish reared under different feeding regimes (r = 0.64) and temperature conditions (r = 0.64). On the dynamics of hIGF-I gene expression in response to elevated temperature, hIGF-I mRNA levels were significantly elevated after at least 2 days of exposure to warm temperature. This validates the concept that hIGF-I gene expressions are sufficiently sensitive to be used as a rapid growth rate indicator for O. niloticus. The hIGF-I levels have a significant positive correlation with specific growth rate (length; r = 0.92), and with condition factor (r = 0.55). On the effect of social stress, differential alterations in growth rates between the dominant and subordinates were observed which was attributed more to behavioral changes as transduced by physiological regulators. The fish's relative position in the social hierarchy was consistently reflected in the levels of hIGF-I mRNA and the eye color pattern. Subordination depressed hIGF-I levels while dominance stimulated it. These findings have shown that hGF-I level remained positively correlated to growth rate as affected by feeding regime, temperature and social stress. This suggests that hIGF-I plays a key role in controlling growth in O. niloticus and indicates that IGF-I mRNA quantification could prove useful for the rapid assessment of growth rate in this species of fish.

Insulin-Like Growth Factor-I (IGF-1), IGF-Binding Protein-3 (IGFBP-3) and mammographic features

Introduction. The IGF system has recently been shown to play an important role in the regulation of breast tumor cell proliferation. However, also breast density is currently considered as the strongest breast cancer risk factor. It is not yet clear whether these factors are interrelated and if and how they are influenced by menopausal status. The purpose of this study was to examine the possible effects of IGF-1 and IGFBP-3 and IGF-1/IGFBP-3 molar ratio on mammographic density stratified by menopausal status. Patients and methods. A group of 341 Italian women were interviewed to collect the following data: family history of breast cancer, reproductive and menstrual factors, breast biopsies, previous administration of hormonal contraceptive therapy, hormone replacement therapy (HRT) in menopause and lifestyle information. A blood sample was drawn for determination of IGF-1, IGFBP-3 levels. IGF-1/ IGFBP-3 molar ratio was then calculated. On the basis of recent mammograms the women were divided into two groups: dense breast (DB) and non-dense breast (NDB). Student’s t-test was employed to assess the association between breast density and plasma level of IGF-1, IGFBP-3 and molar ratio. To assess if this relationship was similar in subgroups of pre- and postmenopausal women, the study population was stratified by menopausal status and Student’s t-test was performed. Finally, multivariate analysis was employed to evaluate if there were confounding factors that might influence the relationship between growth factors and breast density. Results. The analysis of the relationship between mammographic density and plasma level of IGF-1, IGFBP-3 and IGF-1/ IGFBP-3 molar ratio showed that IGF-1 levels and molar ratio varied in the two groups resulting in higher mean values in the DB group (IGF-1: 109.6 versus 96.6 ng/ml; p= 0.001 and molar ratio 29.4 versus 25.5 ng/ml; p= 0.001) whereas IGFBP-3 showed similar values in both groups (DB and NDB). Analysis of plasma level of IGF-1, IGFBP-3 and IGF-1/IGFBP-3 molar ratio compared to breast density after stratification of the study population by menopausal status (premenopausal and postmenopausal) showed that there was no association between the plasma of growth factors and breast density, neither in premenopausal nor in postmenopausal patients. Multivariate analysis showed that only nulliparity, premenopausal status and body mass index (BMI) are determinants of breast density. Conclusions. Our study provides a strong evidence of a crude association between breast density and plasma levels of IGF-1 and molar ratio. On the basis of our results, it is reasonable to assume that the role of IGF-1 and molar ratio in the pathogenesis of breast cancer might be mediated through mammographic density. IGF-1 and molar ratio might thus increase the risk of cancer by increasing mammographic density.

Hypoxia induces chondrocyte-specific gene expression in mesenchymal cells in association with transcriptional activation of Sox9

Endochondral bone is formed during an avascular period in an environment of low oxygen. Under these conditions, pluripotential mesenchymal stromal cells preferentially differentiate into chondrocytes and form cartilage. In this study, we investigated the hypothesis that oxygen tension modulates bone mesenchymal cell fate by altering the expression of genes that function to promote chondrogenesis. Microarray of RNA samples from ST2 cells revealed significant changes in 728 array elements (P < 0.01) in response to hypoxia. Real-time PCR on these RNA samples, and separate samples from C3H10T1/2 cells, revealed hypoxia-induced changes in the expression of additional genes known to be expressed by chondrocytes including Sox9 and its downstream targets aggrecan and Col2a. These changes were accompanied by the accumulation of mucopolysacharide as detected by alcian blue staining. To investigate the mechanisms responsible for upregulation of Sox9 by hypoxia, we determined the effect of hypoxia on HIF-1 alpha levels and Sox9 promoter activity in ST2 cells. Hypoxia increased nuclear accumulation of HIF-1 alpha and activated the Sox9 promoter. The ability of hypoxia to transactivate the Sox9 promoter was virtually abolished by deletion of HIF-1 alpha consensus sites within the proximal promoter. These findings suggest that hypoxia promotes the differentiation of mesenchymal cells along a chondrocyte pathway in part by activating Sox-9 via a HIF-1 alpha-dependent mechanism. (c) 2005 Elsevier Inc. All rights reserved.

The human granulocyte-macrophage colony- stimulating factor gene is autonomously regulated in vivo by an inducible tissue-specific enhancer

The granulocyte-macrophage colony-stimulating factor (GM-CSF) gene is part of a cytokine gene cluster and is directly linked to a conserved upstream inducible enhancer. Here we examined the in vitro and in vivo functions of the human GM-CSF enhancer and found that it was required for the correctly regulated expression of the GM-CSF gene. An inducible DNase I-hypersensitive site appeared within the enhancer in cell types such as T cells, myeloid cells, and endothelial cells that express GM-CSF, but not in nonexpressing cells. In a panel of transfected cells the human GM-CSF enhancer was activated in a tissue-specific manner in parallel with the endogenous gene. The in vivo function of the enhancer was examined in a transgenic mouse model that also addressed the issue of whether the GM-CSF locus was correctly regulated in isolation from other segments of the cytokine gene cluster. After correction for copy number the mean level of human GM-CSF expression in splenocytes from 11 lines of transgenic mice containing a 10.5-kb human GM-CSF transgene was indistinguishable from mouse GM-CSF expression (99% ± 56% SD). In contrast, a 9.8-kb transgene lacking just the enhancer had a significantly reduced (P = 0.004) and more variable level of activity (29% ± 89% SD). From these studies we conclude that the GM-CSF enhancer is required for the correct copy number-dependent expression of the human GM-CSF gene and that the GM-CSF gene is regulated independently from DNA elements associated with the closely linked IL-3 gene or other members of the cytokine gene cluster.

Origin and evolution of the RIG-I like RNA helicase gene family

Background: The DExD/H domain containing RNA helicases such as retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are key cytosolic pattern recognition receptors (PRRs) for detecting nucleotide pathogen associated molecular patterns (PAMPs) of invading viruses. The RIG-I and MDA5 proteins differentially recognise conserved PAMPs in double stranded or single stranded viral RNA molecules, leading to activation of the interferon system in vertebrates. They share three core protein domains including a RNA helicase domain near the C terminus (HELICc), one or more caspase activation and recruitment domains (CARDs) and an ATP dependent DExD/H domain. The RIG-I/MDA5 directed interferon response is negatively regulated by laboratory of genetics and physiology 2 (LGP2) and is believed to be controlled by the mitochondria antiviral signalling protein (MAVS), a CARD containing protein associated with mitochondria. Results: The DExD/H containing RNA helicases including RIG-I, MDA5 and LGP2 were analysed in silico in a wide spectrum of invertebrate and vertebrate genomes. The gene synteny of MDA5 and LGP2 is well conserved among vertebrates whilst conservation of the gene synteny of RIG-I is less apparent. Invertebrate homologues had a closer phylogenetic relationship with the vertebrate RIG-Is than the MDA5/LGP2 molecules, suggesting the RIG-I homologues may have emerged earlier in evolution, possibly prior to the appearance of vertebrates. Our data suggest that the RIG-I like helicases possibly originated from three distinct genes coding for the core domains including the HELICc, CARD and ATP dependent DExD/H domains through gene fusion and gene/domain duplication. Furthermore, presence of domains similar to a prokaryotic DNA restriction enzyme III domain (Res III), and a zinc finger domain of transcription factor (TF) IIS have been detected by bioinformatic analysis. Conclusion: The RIG-I/MDA5 viral surveillance system is conserved in vertebrates. The RIG-I like helicase family appears to have evolved from a common ancestor that originated from genes encoding different core functional domains. Diversification of core functional domains might be fundamental to their functional divergence in terms of recognition of different viral PAMPs.

Diversities in hepatic HIF-1, IGF-I/IGFBP-1, LDH/ICD, and their mRNA expressions induced by CoCl2 in Qinghai-Tibetan plateau mammals and sea level mice

Ochotona curzoniae and Microtus oeconomus are the native mammals living on the Qinghai-TibetanPlateau of China. The molecular mechanisms of their acclimatization to the Plateau-hypoxia remain unclear. Expressions of hepatic hypoxia-inducible factor (HIF)-1 alpha, insulin-like growth factor-I (IGF-I)/IGF binding protein (BP)-1(IGFBP-1; including genes), and key metabolic enzymatic genes [lactate dehydrogenase (LDH)-A/isocitrate dehydrogenase (ICD)] are compared in Qinghai-Tibetan- Plateau mammals andsea- level mice after injection of CoCl2 (20, 40, or 60 mg/ kg) and normobaric hypoxia (16.0% O-2, 10.8% O-2, and 8.0% O-2) for 6 h, tested by histochemistry, Western blot analysis, ELISA, and RT-PCR. Major results are CoCl2 markedly increased 1) HIF-1 alpha only in mice, 2) hepatic and circulatory IGF-I in M. oeconomus, 3) hepatic IGFBP-1 in mice and O. curzoniae, and 4) LDH-A but reduced ICD mRNA in mice (CoCl2 20 mg/kg) but were unchanged in the Tibetan mammals. Normobaric hypoxia markedly 1) increased HIF-1 alpha and LDH-A mRNA in mice and M. oeconomus (8.0% O-2) not in O. curzoniae, and 2) reduced ICD mRNA in mice and M. oeconomus (8.0% O-2) not in O. curzoniae. Results suggest that 1) HIF-1 alpha responsiveness to hypoxia is distinct in lowland mice and plateau mammals, reflecting a diverse tolerance of the three species to hypoxia; 2) CoCl2 induces diversities in HIF-1, IGF-I/IGFBP-1 protein or genes in mice, M. oeconomus, and O. curzoniae. In contrast, HIF-1 mediates IGFBP-1 transcription only in mice and in M. oeconomus (subjected to severe hypoxia); 3) differences in IGF-I/IGFBP-1 expressions induced by CoCl2 reflect significant diversities in hormone regulation and cell protection from damage; and 4) activation of anaerobic glycolysis and reduction of Krebs cycle represents strategies of lowland-animals vs. the stable metabolic homeostasis of plateau- acclimatized mammals.

Exploitation of Ebola Virus VP35 Protein to identify new drugs counteracting its type I IFN antagonism

Ebolaviruses (EBOVs) are among the most virulent and deadly pathogens ever known, causing fulminant haemorrhagic fevers in humans and non-human primates. The 2014 outbreak of Ebola virus disease (EVD) in West Africa has claimed more lives than all previous EVD outbreaks combined. The EBOV high mortality rates have been related to the virus-induced impairment of the host innate immunity reaction due to two virus-coded proteins, VP24 and VP35. EBOV VP35 is a multifunctional protein, it is essential for viral replication as a component of the viral RNA polymerase and it also participates in nucleocapsid assembly. Early during EBOV infection, alpha-beta interferon (IFN-α/β) production would be triggered upon recognition of viral dsRNA products by cytoplasmic retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs). However, this recognition is efficiently prevented by the double-stranded RNA (dsRNA) binding activity of the EBOV VP35 protein, which hides RLRs binding sites on the dsRNA phosphate backbone as well the 5’-triphosphate (5’-ppp) dsRNA ends to RIG-I recognition. In addition to dsRNA binding and sequestration, EBOV VP35 inhibits IFN-α/β production preventing the activation of the IFN regulatory factor 3 (IRF-3) by direct interaction with cellular proteins. Previous studies demonstrated that single amino acid changes in the VP35 dsRNA binding domain reduce EBOV virulence, indicating that VP35 is an attractive target for antiviral drugs development. Within this context, here we report the establishment of a novel method to characterize the EBOV VP35 inhibitory function of the dsRNA-dependent RIG-I-mediated IFN-β signaling pathway in a BLS2 cell culture setting. In such system, a plasmid containing the promoter region of IFN-β gene linked with a luciferase reporter gene was transfected, together with a EBOV VP35 mammalian expression plasmid, into the IFN-sensitive A549 cell line, and the IFN-induction was stimulated through dsRNA transfection. Through alanine scanning mutational studies with biochemical, cellular and computational methods we highlighted the importance of some VP35 residues involved in dsRNA end-capping binding, such as R312, K282 and R322, that may serve as target for the development of small-molecule inhibitors against EBOV. Furthermore, we identified a synthetic compound that increased IFN-induction only under antiviral response stimulation and subverted VP35 inhibition, proving to be very attractive for the development of an antiviral drug. In conclusion, our results provide the establishment of a new assay as a straightforward tool for the screening of antiviral compounds that target i) dsRNA-VP35 or cellular protein-VP35 interaction and ii) dsRNA-dependent RIG-I-mediated IFN signaling pathway, in order to potentiate the IFN response against VP35 inhibition, setting the bases for further drug development.

Modulation of hypoxia-inducible factors as a therapeutic target for multiple myeloma

Hypoxia-inducible factor-1 alpha (HIF-1α) plays a critical role in survival and is associated with poor prognosis in solid tumors. The role of HIF-1α in multiple myeloma is not completely known. In the present study, we explored the effect of EZN2968, an locked nucleic acid antisense oligonucleotide against HIF-1α, as a molecular target in MM. A panel of MM cell lines and primary samples from MM patients were cultured in vitro in the presence of EZN2968 . Under normoxia culture condition, HIF-1α mRNA and protein expression was detectable in all MM cell lines and in CD138+ cells from newly diagnosed MM patients samples. Significant up-regulation of HIF-1α protein expression was observed after incubation with IL6 or IGF-I, confirming that HIF-1α can be further induced by biological stimuli. EZN2968 efficiently induces a selective and stable down-modulation of HIF-1α and decreased the secretion of VEGF released by MM cell. Treatment with EZN2968 gave rise to a progressive accumulation of cells in the S and subG0 phase. The analysis of p21, a cyclin-dependent kinase inhibitors controlling cell cycle check point, shows upregulation of protein levels. These results suggest that HIF-1α inhibition is sufficient for cell cycle arrest in normoxia, and for inducing an apoptotic pathways.. In the presence of bone marrow microenvironment, HIF-1α inhibition blocks MAPK kinase pathway and secretion of pro-surviaval cytokines ( IL6,VEGF,IL8) In this study we provide evidence that HIF-1α, even in the absence of hypoxia signal, is expressed in MM plasma cells and further inducible by bone marrow milieu stimuli; moreover its inhibition is sufficient to induce a permanent cell cycle arrest. Our data support the hypothesis that HIF-1α inhibition may suppress tumor growth by preventing proliferation of plasma cells through p21 activation and blocking pro-survival stimuli from bone marrow microenvironment.

Canine keratinocytes upregulate type I interferons and proinflammatory cytokines in response to poly(dA:dT) but not to canine papillomavirus.

Papillomaviruses (PV) are double stranded (ds) DNA viruses that infect epithelial cells within the skin or mucosa, most often causing benign neoplasms that spontaneously regress. The immune system plays a key role in the defense against PVs. Since these viruses infect keratinocytes, we wanted to investigate the role of the keratinocyte in initiating an immune response to canine papillomavirus-2 (CPV-2) in the dog. Keratinocytes express a variety of pattern recognition receptors (PRR) to distinguish different cutaneous pathogens and initiate an immune response. We examined the mRNA expression patterns for several recently described cytosolic nucleic acid sensing PRRs in canine monolayer keratinocyte cultures using quantitative reverse transcription-polymerase chain reaction. Unstimulated normal cells were found to express mRNA for melanoma differentiation associated gene 5 (MDA5), retinoic acid-inducible gene I (RIG-I), DNA-dependent activation of interferon regulatory factors, leucine rich repeat flightless interacting protein 1, and interferon inducible gene 16 (IFI16), as well as their adaptor molecules myeloid differentiation primary response gene 88, interferon-β promoter stimulator 1, and endoplasmic reticulum-resident transmembrane protein stimulator of interferon genes. When stimulated with synthetic dsDNA [poly(dA:dT)] or dsRNA [poly(I:C)], keratinocytes responded with increased mRNA expression levels for interleukin-6, tumor necrosis factor-α, interferon-β, RIG-I, IFI16, and MDA5. There was no detectable increase in mRNA expression, however, in keratinocytes infected with CPV-2. Furthermore, CPV-2-infected keratinocytes stimulated with poly(dA:dT) and poly(I:C) showed similar mRNA expression levels for these gene products when compared with expression levels in uninfected cells. These results suggest that although canine keratinocytes contain functional PRRs that can recognize and respond to dsDNA and dsRNA ligands, they do not appear to recognize or initiate a similar response to CPV-2.