Increased Frataxin Expression Induced in Friedreich Ataxia Cells by Platinum TALE-VP64s or Platinum TALE-SunTag.

Frataxin gene (FXN) expression is reduced in Friedreich's ataxia patients due to an increase in the number of GAA trinucleotides in intron 1. The frataxin protein, encoded by that gene, plays an important role in mitochondria's iron metabolism. Platinum TALE (plTALE) proteins targeting the regulatory region of the FXN gene, fused with a transcriptional activator (TA) such as VP64 or P300, were used to increase the expression of that gene. Many effectors, plTALEVP64, plTALEp300, and plTALESunTag, targeting 14 sequences of the FXN gene promoter or intron 1 were produced. This permitted selection of 3 plTALEVP64s and 2 plTALESunTag that increased FXN gene expression by up to 19-fold in different Friedreich ataxia (FRDA) primary fibroblasts. Adeno-associated viruses were used to deliver the best effectors to the YG8R mouse model to validate their efficiencies in vivo. Our results showed that these selected plTALEVP64s or plTALESunTag induced transcriptional activity of the endogenous FXN gene as well as expression of the frataxin protein in YG8R mouse heart by 10-fold and in skeletal muscles by up to 35-fold. The aconitase activity was positively modulated by the frataxin level in mitochondria, and it was, thus, increased in vitro and in vivo by the increased frataxin expression.

Efficient Restoration of the Dystrophin Gene Reading Frame and Protein Structure in DMD Myoblasts Using the CinDel Method.

The CRISPR/Cas9 system is a great revolution in biology. This technology allows the modification of genes in vitro and in vivo in a wide variety of living organisms. In most Duchenne muscular dystrophy (DMD) patients, expression of dystrophin (DYS) protein is disrupted because exon deletions result in a frame shift. We present here the CRISPR-induced deletion (CinDel), a new promising genome-editing technology to correct the DMD gene. This strategy is based on the use of two gRNAs targeting specifically exons that precede and follow the patient deletion in the DMD gene. This pair of gRNAs induced a precise large additional deletion leading to fusion of the targeted exons. Using an adequate pair of gRNAs, the deletion of parts of these exons and the intron separating them restored the DMD reading frame in 62% of the hybrid exons in vitro in DMD myoblasts and in vivo in electroporated hDMD/mdx mice. Moreover, adequate pairs of gRNAs also restored the normal spectrin-like repeat of the dystrophin rod domain; such restoration is not obtained by exon skipping or deletion of complete exons. The expression of an internally deleted DYS protein was detected following the formation of myotubes by the unselected, treated DMD myoblasts. Given that CinDel induces permanent reparation of the DMD gene, this treatment would not have to be repeated as it is the case for exon skipping induced by oligonucleotides.

Generation of human endometrial knockout cell lines with the CRISPR/Cas9 system confirms the prostaglandin F2α synthase activity of aldo-ketoreductase 1B1.

Prostaglandins (PGs) are important regulators of female reproductive function. The primary PGs produced in the endometrium are PGE2 and PGF2α. Relatively little is known about the biosynthetic pathways leading to the formation of PGF2α. We have described the role of aldo-ketoreductase (AKR)1B1 in increased PGF2α production by human endometrial cells following stimulation with interleukin-1ß (IL-1ß). However, alternate PGF synthases are expressed concurrently in endometrial cells. A definite proof of the role of AKR1B1 would require gene knockout; unfortunately, this gene has no direct equivalent in the mouse. Recently, an efficient genome-editing technology using RNA-guided DNase Cas9 and the clustered regularly interspaced short palindromic repeats (CRISPR) system has been developed. We have adapted this approach to knockout AKR1B1 gene expression in human endometrial cell lines. One clone (16-2) of stromal origin generated by the CRISPR/Cas9 system exhibited a complete loss of AKR1B1 protein and mRNA expression, whereas other clones presented with partial edition. The present report focuses on the characterization of clone 16-2 exhibiting deletion of 68 and 2 nucleotides, respectively, on each of the alleles. Cells from this clone lost their ability to produce PGF2α but maintained their original stromal cell (human endometrial stromal cells-2) phenotype including the capacity to decidualize in the presence of progesterone (medroxyprogesterone acetate) and 8-bromo-cAMP. Knockout cells also maintained their ability to increase PGE2 production in response to IL-1ß. In summary, we demonstrate that the new genome editing CRISPR/Cas9 system can be used in human cells to generate stable knockout cell line models. Our results suggest that genome editing of human cell lines can be used to complement mouse KO models to validate the function of genes in differentiated tissues and cells. Our results also confirm that AKR1B1 is involved in the synthesis of PGF2α.

An AAV9 coding for frataxin clearly improved the symptoms and prolonged the life of Friedreich ataxia mouse models.

Friedreich ataxia (FRDA) is a genetic disease due to increased repeats of the GAA trinucleotide in intron 1 of the frataxin gene. This mutation leads to a reduced expression of frataxin. We have produced an adeno-associated virus (AAV)9 coding for human frataxin (AAV9-hFXN). This AAV was delivered by intraperitoneal (IP) injection to young conditionally knockout mice in which the frataxin gene had been knocked-out in some tissues during embryogenesis by breeding them with mice expressing the Cre recombinase gene under the muscle creatine kinase (MCK) or the neuron-specific enolase (NSE) promoter. In the first part of the study, different doses of virus were tested from 6 × 10(11) v.p. to 6 × 10(9) v.p. in NSE-cre mice and all leading to an increase in life spent of the mice. The higher and the lower dose were also tested in MCK-cre mice. A single administration of the AAV9-hFXN at 6 × 10(11) v.p. more than doubled the life of these mice. In fact the MCK-cre mice treated with the AAV9-hFXN were sacrificed for further molecular investigations at the age of 29 weeks without apparent symptoms. Echography analysis of the heart function clearly indicated that the cardiac systolic function was better preserved in the mice that received 6 × 10(11) v.p. of AAV9-hFXN. The human frataxin protein was detected by ELISA in the heart, brain, muscles, kidney, and liver with the higher dose of virus in both mouse models. Thus, gene therapy with an AAV9-hFXN is a potential treatment of FRDA.

A Potential New Therapeutic Approach for Friedreich Ataxia: Induction of Frataxin Expression With TALE Proteins.

TALEs targeting a promoter sequence and fused with a transcription activation domain (TAD) may be used to specifically induce the expression of a gene as a potential treatment for haploinsufficiency. This potential therapeutic approach was applied to increase the expression of frataxin in fibroblasts of Friedreich ataxia (FRDA) patients. FRDA fibroblast cells were nucleofected with a pCR3.1 expression vector coding for TALEFrat#8 fused with VP64. A twofold increase of the frataxin mRNA (detected by quantitative reverse transcription-PCR (qRT-PCR)) associated with a similar increase of the mature form of the frataxin protein was observed. The frataxin mRNA and protein were also increased by this TALE in the fibroblasts of the YG8R mouse model. The addition of 5-aza-2'-deoxycytidine (5-Aza-dC) or of valproic acid (VPA) to the TALE treatment did not produce significant improvement. Other TADs (i.e., p65, TFAP2α, SRF, SP1, and MyoD) fused with the TALEFrat#8 gene did not produce a significant increase in the frataxin protein. Thus the TALEFrat#8-VP64 recombinant protein targeting the frataxin promoter could eventually be used to increase the frataxin expression and alleviate the FRDA symptoms.Molecular Therapy-Nucleic Acids (2013) 2, e119; doi:10.1038/mtna.2013.41; published online 3 September 2013.

Evaluation of the prostaglandin F synthase activity of human and bovine aldo-keto reductases: AKR1A1s complement AKR1B1s as potent PGF synthases.

AKR1B1 of the polyol pathway was identified as a prostaglandin F2α synthase (PGFS). Using a genomic approach we have identified in the endometrium five bovine and three human AKRs with putative PGFS activity and generated the corresponding recombinant enzymes. The PGFS activity of the recombinant proteins was evaluated using a novel assay based on in situ generation of the precursor of PG biosynthesis PGH2. PGF2α was measured by ELISA and the relative potencies of the different enzymes were compared. We identified AKR1A1 and confirmed AKR1B1 as the most potent PGFS expressing characteristic inhibition patterns in presence of methylglyoxal, ponalrestat and glucose.

Targeted Gene Addition of Microdystrophin in Mice Skeletal Muscle via Human Myoblast Transplantation.

Zinc finger nucleases (ZFN) can facilitate targeted gene addition to the genome while minimizing the risks of insertional mutagenesis. Here, we used a previously characterized ZFN pair targeting the chemokine (C-C motif) receptor 5 (CCR5) locus to introduce, as a proof of concept, the enhanced green fluorescent protein (eGFP) or the microdystrophin genes into human myoblasts. Using integrase-defective lentiviral vectors (IDLVs) and chimeric adenoviral vectors to transiently deliver template DNA and ZFN respectively, we achieved up to 40% targeted gene addition in human myoblasts. When the O(6)-methylguanine-DNA methyltransferase(P140K) gene was co-introduced with eGFP, the frequency of cells with targeted integration could be increased to over 90% after drug selection. Importantly, gene-targeted myoblasts retained their mitogenic activity and potential to form myotubes both in vitro and in vivo when injected into the tibialis anterior of immune-deficient mice. Altogether, our results could lead to the development of improved cell therapy transplantation protocols for muscular diseases.Molecular Therapy - Nucleic Acids (2013) 2, e68; doi:10.1038/mtna.2012.55; published online 29 January 2013.

The Prostaglandin F Synthase Activity of the Human Aldose Reductase AKR1B1 Brings New Lenses to Look at Pathologic Conditions.

Prostaglandins are important regulators of female reproductive functions to which aldose reductases exhibiting hydroxysteroid dehydrogenase activity also contribute. Our work on the regulation of reproductive function by prostaglandins (PGs), lead us to the discovery that AKR1B5 and later AKR1B1were highly efficient and physiologically relevant PGF synthases. PGE2 and PGF2α are the main prostanoids produced in the human endometrium and proper balance in their relative production is important for normal menstruation and optimal fertility. Recent evidence suggests that PGE2/EP2 and PGF2α/FP may constitute a functional dyad with physiological relevance comparable to the prostacyclin-thromboxane dyad in the vascular system. We have recently reported that AKR1B1 was expressed and modulated in association with PGF2α production in response to IL-1ß in the human endometrium. In the present study, we show that the human AKR1B1 (gene ID: 231) also known as ALDR1 or ALR2 is a functional PGF2α synthase in different models of living cells and tissues. Using human endometrial cells, prostate, and vascular smooth muscle cells, cardiomyocytes and endothelial cells we demonstrate that IL-1ß is able to up regulate COX-2 and AKR1B1 proteins as well as PGF2α production under normal glucose concentrations. We show that the promoter activity of AKR1B1 gene is increased by IL-1ß particularly around the multiple stress response region containing two putative antioxidant response elements adjacent to TonE and AP1. We also show that AKR1B1 is able to regulate PGE2 production through PGF2α acting on its FP receptor and that aldose reductase inhibitors like alrestatin, Statil (ponalrestat), and EBPC exhibit distinct and characteristic inhibition of PGF2α production in different cell models. The PGF synthase activity of AKR1B1 represents a new and important target to regulate ischemic and inflammatory responses associated with several human pathologies.

Transcription activator-like effector proteins induce the expression of the frataxin gene.

Genes encoding transcription activator-like effector (TALE) proteins may be engineered to target specific DNA sequences. TALEs fused with a transcription activator can be used to specifically induce the expression of a gene. This could lead to completely new therapies for several diseases. We have applied this potential therapeutic approach to Friedreich ataxia (FRDA), as an example. FRDA is due to reduced expression of frataxin because of elongation of a trinucleotide (GAA) repeat in intron 1. Our aim was to develop a potential treatment for FRDA by increasing the expression of the frataxin gene. We engineered 12 TALE genes (TALE(Frat)) encoding TALE(Frat) proteins, each specifically targeting different 14-bp DNA sequences within the proximal region of the human frataxin promoter. When the genes encoding these TALE(Frat) proteins were fused with a transcription activator, that is, four VP16 peptides (i.e., VP64), the resulting TALE(Frat)-VP64 proteins induced the expression of an mCherry reporter gene fused to a mini-cytomegalovirus promoter able to be activated by the insertion of the frataxin proximal promoter upstream to the minipromoter. These TALE(Frat)-VP64 proteins also increased, by 2- to 3-fold, frataxin gene expression (detected by qRT-PCR) in the cells. We conclude that TALE(Frat) proteins targeting the frataxin promoter may be used to increase the expression of frataxin mRNA and potentially could alleviate the symptoms of Friedreich ataxia. TALE methodology opens a new field of research, which could be used to develop TALE proteins to treat other diseases by inducing the expression of specific genes.

Abnormal prostaglandin E2 production blocks myogenic differentiation in myotonic dystrophy.

The congenital form of myotonic dystrophy type 1 (DM1) is the most severe type of the disease associated with CTG expansions over 1500 repeats and delayed muscle maturation. The mechanistic basis of the congenital form of DM1 is mostly unknown. Here, we show that muscle satellite cells bearing large CTG expansions (>3000) secrete a soluble factor that inhibits the fusion of normal myoblasts in culture. We identified this factor as prostaglandin E2 (PGE(2)). In these DM1 cells, PGE(2) production is increased through up-regulation of cyclooxygenase 2 (Cox-2), mPGES-1 and prostaglandin EP2/EP4 receptors. Elevated levels of PGE(2) inhibit myogenic differentiation by decreasing the intracellular levels of calcium. Exogenous addition of acetylsalicylic acid, an inhibitor of Cox enzymes, abolishes PGE(2) abnormal secretion and restores the differentiation of DM1 muscle cells. These data indicate that the delay in muscle maturation observed in congenital DM1 may result, at least in part, from an altered autocrine mechanism. Inhibitors of prostaglandin synthesis may thus offer a powerful method to restore the differentiation of DM1 muscle cells.

The multidrug resistance-associated protein 4 (MRP4) appears as a functional carrier of prostaglandins regulated by oxytocin in the bovine endometrium.

Prostaglandins (PG) are involved in several female reproductive processes, and their action is regulated at the levels of biosynthesis, catabolism, and signal transduction. Facilitated transport across cell membranes emerges as an additional checkpoint regulating PG action. We have already reported on the influx transporter solute carrier organic anion transporting polypeptide (SLCO2A1) [PG transporter (PGT)] in relation to PG action in the bovine endometrium. In the present study, we report on the functional expression and regulation of multidrug resistance-associated protein 4 (MRP4)/ATP-binding cassette carrier 4, an alternate PG transporter belonging to the ATP-binding cassette carrier (ABC) family. We have found that MRP4 protein was present throughout the estrous cycle and exhibited a pattern of expression similar to that of PGT with maximal expression during early-mid luteal phase in the bovine endometrium. Functional expression and regulation of MRP4 was studied in vitro using the newly developed bovine endometrial epithelial bEEL and stromal CSC cell lines. Oxytocin (OT) stimulated PGF2α production and MRP4 mRNA and protein in a time- and dose-dependent manner but had no effect on PGT. OT induced preferred accumulation of PG outside the cells and secretion toward the basolateral side of polarized bEEL cells grown on membrane inserts. MK-571 and indomethacin, two documented inhibitors of MRP4 activity, blocked preferred accumulation of PG, but interferon-τ and NS-398 had no effect on MRP4 expression or the direction of PG transport. Our results suggest that MRP4 is a functional PG carrier under the regulation of OT in the bovine endometrium.

Endonucleases: tools to correct the dystrophin gene.

BACKGROUND: Various endonucleases can be engineered to induce double-strand breaks (DSBs) in chosen DNA sequences. These DSBs are spontaneously repaired by nonhomologous-end-joining, resulting in micro-insertions or micro-deletions (INDELs). We detected, characterized and quantified the frequency of INDELs produced by one meganuclease (MGN) targeting the RAG1 gene, six MGNs targeting three introns of the human dystrophin gene and one pair of zinc finger nucleases (ZFNs) targeting exon 50 of the human dystrophin gene. The experiments were performed in human cells (i.e. 293 T cells, myoblasts and myotubes). METHODS: To analyse the INDELs produced by the endonucleases the targeted region was polymerase chain reaction amplified and the amplicons were digested with the Surveyor enzyme, cloned in bacteria or deep sequenced. RESULTS: Endonucleases targeting the dystrophin gene produced INDELs of different sizes but there were clear peaks in the size distributions. The positions of these peaks were similar for MGNs but not for ZFNs in 293 T cells and in myoblasts. The size of the INDELs produced by these endonucleases in the dystrophin gene would have permitted a change in the reading frame. In a subsequent experiment, we observed that the frequency of INDELs was increased by re-exposition of the cells to the same endonuclease. CONCLUSIONS: Endonucleases are able to: (i) restore the normal reading of a gene with a frame shift mutation; (ii) delete a nonsense codon; and (iii) knockout a gene. Endonucleases could thus be used to treat Duchenne muscular dystrophy and other hereditary diseases that are the result of a nonsense codon or a frame shift mutation.

The human aldose reductase AKR1B1 qualifies as the primary prostaglandin F synthase in the endometrium.

CONTEXT: Prostaglandins (PGs) E2 and PGF2α are produced in the endometrium and are important for menstruation and fertility. Dysmenorrhea is associated with increased production of PGF2α relative to PGE2, and the opposite is true for menorrhagia. The pathways leading to PGE2 biosynthesis are well described, but little is known for PGF2α. Aldoketoreductase (AKR)-1C3, the only PGF synthase identified in the human, cannot explain the production of PGF2α by endometrial cells. AKR1B1 appears to be an alternate candidate with promising therapeutic value. OBJECTIVE: The objective of the study was to address whether AKR1B1 (gene ID 231) is a functional PGF2α synthase in the human endometrium and a valid therapeutic target for menstrual pain. DESIGN: The design of the study was basic laboratory analyses to identify gene expression and protein levels associated with PGF2α production in endometrial tissues and endometrial cells from cycling women aged between 23 and 52 yr undergoing biopsies or hysterectomy for diverse gynecological disorders. RESULTS: AKR1B1 is expressed at a high level during the menstrual cycle during the secretory phase and in both epithelial and stromal cells, whereas AKR1C3 was found only in epithelial cells. Purified recombinant AKR1B1 protein, gene silencing, and transient transfection experiments all concur to demonstrate that this enzyme is a functional PGF synthase. Ponalrestat, a specific inhibitor developed to block AKR1B1 activity, reduced PGF2α production in response to IL-1ß in both cultured endometrial cells and endometrial explants. CONCLUSIONS: The human aldose reductase AKR1B1 currently associated with diabetes complications is also a highly functional PGF synthase responsible for PGF2α production in the human endometrium and a potential target for treatment of menstrual disorders.

Intramuscular transplantation of human postnatal myoblasts generates functional donor-derived satellite cells.

Myogenic cell transplantation is an experimental approach for the treatment of myopathies. In this approach, transplanted cells need to fuse with pre-existing myofibers, form new myofibers, and generate new muscle precursor cells (MPCs). The last property was fully reported following myoblast transplantation in mice but remains poorly studied with human myoblasts. In this study, we provide evidence that the intramuscular transplantation of postnatal human myoblasts in immunodeficient mice generates donor-derived MPCs and specifically donor-derived satellite cells. In a first experiment, cells isolated from mouse muscles 1 month after the transplantation of human myoblasts proliferated in vitro as human myoblasts. These cells were retransplanted in mice and formed myofibers expressing human dystrophin. In a second experiment, we observed that inducing muscle regeneration 2 months following transplantation of human myoblasts led to myofiber regeneration by human-derived MPCs. In a third experiment, we detected by immunohistochemistry abundant human-derived satellite cells in mouse muscles 1 month after transplantation of postnatal human myoblasts. These human-derived satellite cells may correspond totally or partially to the human-derived MPCs evidenced in the first two experiments. Finally, we present evidence that donor-derived satellite cells may be produced in patients that received myoblast transplantation.

Electrotransfer of the full-length dog dystrophin into mouse and dystrophic dog muscles.

Duchenne muscular dystrophy (DMD) is an X-linked genetic disease characterized by the absence of dystrophin (427 kDa). An approach to eventually restore this protein in patients with DMD is to introduce into their muscles a plasmid encoding dystrophin cDNA. Because the phenotype of the dystrophic dog is closer to the human phenotype than is the mdx mouse phenotype, we have studied the electrotransfer of a plasmid carrying the full-length dog dystrophin (FLDYS(dog)) in dystrophic dog muscle. To achieve this nonviral delivery, the FLDYS(dog) cDNA was cloned in two plasmids containing either a cytomegalovirus or a muscle creatine kinase promoter. In both cases, our results showed that the electrotransfer of these large plasmids (∼17 kb) into mouse muscle allowed FLDYS(dog) expression in the treated muscle. The electrotransfer of pCMV.FLDYS(dog) in a dystrophic dog muscle also led to the expression of dystrophin. In conclusion, introduction of the full-length dog dystrophin cDNA by electrotransfer into dystrophic dog muscle is a potential approach to restore dystrophin in patients with DMD. However, the electrotransfer procedure should be improved before applying it to humans.

Expression of dog microdystrophin in mouse and dog muscles by gene therapy.

Duchenne muscular dystrophy (DMD) is characterized by the absence of dystrophin. Several previous studies demonstrated the feasibility of delivering microdystrophin complementary DNA (cDNA) into mouse and normal nonhuman primate muscles by ex vivo gene therapy. However, these animal models do not reproduce completely the human DMD phenotype, while the dystrophic dog model does. To progress toward the use of the best animal model of DMD, a dog microdystrophin was transduced into human and dystrophic dog muscle precursor cells (MPCs) with a lentivirus before their transplantation into mouse muscles. One month following MPC transplantation, myofibers expressing the dog microdystrophin were observed. We also used another approach to introduce this transgene into myofibers, i.e., the electrotransfer of a plasmid coding for the dog microdystrophin. The plasmid was injected into mouse and dog muscles, and brief electric pulses were applied in the region of injection. Two weeks later, the transgene was detected in both animals. Therefore, ex vivo gene therapy and electrotransfer are two possible methods to introduce a truncated version of dystrophin into myofibers of animal models and eventually into myofibers of DMD patients.

Epidermal growth factor receptor is an obligatory intermediate for oxytocin-induced cyclooxygenase 2 expression and prostaglandin F2 alpha production in bovine endometrial epithelial cells.

Oxytocin (OT) triggers the luteolytic pulses of prostaglandin F(2 alpha) (PGF(2 alpha)) from the endometrial epithelial cells in ruminants. We have proposed that the embryonic signal interferon-tau exerts its antiluteolytic effect by disrupting the OT signaling axis. Accordingly, we have attempted to define the signaling pathway of OT-induced PGF(2 alpha) production in the bovine endometrium using our newly characterized epithelial cell line (bEEL). OT receptor was coupled to the classical G alpha(q) pathway as evidenced by calcium release and activation of phospholipase C. Similarly, OT-induced PGF(2 alpha) production was mediated through the canonical ERK1/2 pathway. Because of the importance of receptor and nonreceptor tyrosine kinases in G protein-coupled receptor signaling, we studied the role of epidermal growth factor receptor (EGFR), c-Src, and phosphoinositide 3-kinase (PI3K) on OT-induced PGF(2 alpha) production in association with cyclooxygenase 2 (COX2) expression and ERK1/2 and Akt phosphorylation. The EGFR inhibitor AG1478 (10 microm) nearly abolished basal and OT-induced PGF(2 alpha) production and down-regulated COX2 expression and ERK1/2 phosphorylation. Because the transactivated EGFR can serve as a ligand for the signaling proteins with Src homology 2 (SH2) domain, we hypothesized a role for c-Src and PI3K in OT-induced PGF(2 alpha) production. Inhibitors of c-Src (PP2, 10 microm) and PI3K (LY294002, 25 microm) produced a significant decrease in OT-induced PGF(2 alpha) production and reduced COX2 expression. Also, PP2, but not LY294002, decreased OT-induced ERK1/2 phosphorylation. Because LY294002 did not affect ERK1/2 phosphorylation, but inhibited PGF(2 alpha) production and down-regulated COX2 expression, it is likely that the Akt pathway is also involved in PGF(2 alpha) production. Thus, EGFR may simultaneously activate c-Src and PI3K to amplify the OT signaling to increase the output of PGF(2 alpha) in bEEL cells.

Development and characterization of a simian virus 40 immortalized bovine endometrial stromal cell line.

In ruminants, interferon-tau (IFNtau) is the maternal recognition signal inhibiting prostaglandin (PG) F2alpha production by endometrial epithelial cells and stimulating interferon-stimulated genes in the stroma. Stromal cells mediate the action of progesterone on epithelial cells during pregnancy. Our working hypothesis is that IFNtau acts as a molecular switch that turns on PGE(2) production in endometrial stromal cells while suppressing PGF2alpha production from epithelial cells. In this report we document immortalization and functional characterization of a bovine stromal cell line from the caruncular region of the endometrium [caruncular stromal cell (CSC)]. Primary stromal cells were immortalized by nucleofection with simian virus 40 large T antigen and integrase. The resulting cell line, CSC, expresses stromal cell-specific vimentin, estrogen, and progesterone receptors, and is amenable for transient transfection. Basal and stimulated production of PGE2 is higher than PGF2alpha and associated with cyclooxygenase (COX) 2 expression. Phorbol myristate acetate (PMA) and IFNtau up-regulate COX2 and PG production in a dose-dependent manner. When added together, low concentrations of IFNtau inhibit PMA-induced COX2 expression; whereas this inhibition is lost at high concentrations. Expression of signal transducer and activator of transcription 1 is induced by IFNtau at all concentrations studied but is not modulated by PMA. Because expression of signal transducer and activator of transcription 1 does not exhibit the biphasic response to IFNtau, we investigated the p38 MAPK pathway using the selective inhibitor SB203580. Inhibition of the p38 MAPK pathway abolishes IFNtau action on PG production. In summary, CSC appears as a good stromal cell model for investigating the molecular mechanisms related to IFNtau action and PG production in the bovine.

Oxytocin receptor down-regulation is not necessary for reducing oxytocin-induced prostaglandin F(2alpha) accumulation by interferon-tau in a bovine endometrial epithelial cell line.

Interferon-tau (IFNtau) is the embryonic signal responsible for pregnancy recognition in ruminants. The primary action of IFNtau is believed to be mediated through inhibition of prostaglandin F(2alpha) (PGF(2alpha)) released from the endometrial epithelial cells in response to oxytocin (OT). Our working hypothesis was that the antiluteolytic effect of IFNtau also involved modulation of PG production downstream of OT receptor (OTR) and/or cyclooxygenase 2 (COX2). There is currently no OT-sensitive endometrial cell line to study the molecular mechanisms underlying our hypotheses. Therefore, we established an immortalized bovine endometrial epithelial cell line (bEEL) exhibiting OT response. These cells were cytokeratin positive, expressed steroid receptors, and exhibited preferential accumulation of PGF(2alpha) over PGE(2). The bEEL cells were highly sensitive to OT, showing time- and concentration-dependent increase in COX2 transcript and protein and PGF(2alpha) accumulation. Interestingly, IFNtau (20 ng/ml) significantly reduced OT-induced PGF(2alpha) accumulation, but surprisingly, the effect was not mediated through down-regulation of either OTR or COX2. Rather, IFNtau up-regulated COX2 in a time- and concentration-dependent manner while decreasing OT-induced PG accumulation. This suggests that COX2 is not a primary target for the antiluteolytic effect of IFNtau. Because IFNtau reduced OT-stimulated PGF(2alpha) accumulation within 3 h, the mechanism likely involves a direct interference at the level of the OT signaling or transcription in addition to the down-regulation of OTR observed in vivo. In summary, bEEL cells offer a unique in vitro model for investigating the cellular and molecular mechanisms underlying OT and IFNtau response in relation with luteolysis and recognition of pregnancy in the bovine.