The Generation of a Comprehensive Spectral Library for the Analysis of the Guinea Pig Proteome by SWATH-MS.

Advances in liquid chromatography-mass spectrometry have facilitated the incorporation of proteomic studies to many biology experimental workflows. Data-independent acquisition platforms, such as sequential window acquisition of all theoretical mass spectra (SWATH-MS), offer several advantages for label-free quantitative assessment of complex proteomes over data-dependent acquisition (DDA) approaches. However, SWATH data interpretation requires spectral libraries as a detailed reference resource. The guinea pig (Cavia porcellus) is an excellent experimental model for translation to many aspects of human physiology and disease, yet there is limited experimental information regarding its proteome. To overcome this knowledge gap, a comprehensive spectral library of the guinea pig proteome is generated. Homogenates and tryptic digests are prepared from 16 tissues and subjected to >200 DDA runs. Analysis of >250 000 peptide-spectrum matches resulted in a library of 73 594 peptides from 7666 proteins. Library validation is provided by i) analyzing externally derived SWATH files (https://doi.org/10.1016/j.jprot.2018.03.023) and comparing peptide intensity quantifications; ii) merging of externally derived data to the base library. This furnishes the research community with a comprehensive proteomic resource that will facilitate future molecular-phenotypic studies using (re-engaging) the guinea pig as an experimental model of relevance to human biology. The spectral library and raw data are freely accessible in the MassIVE repository (MSV000083199).

Cytokine-induced cysteine- serine-rich nuclear protein-1 (CSRNP1) selectively contributes to MMP1 expression in human chondrocytes.

Irreversible cartilage collagen breakdown by the collagenolytic matrix metalloproteinases (MMPs)-1 and MMP-13 represents a key event in pathologies associated with tissue destruction such as arthritis. Inflammation is closely associated with such pathology and occurs in both rheumatoid and osteoarthritis making it highly relevant to the prevailing tissue damage that characterises these diseases. The inflammation-induced activating protein-1 (AP-1) transcription factor is an important regulator of both MMP1 and MMP13 genes with interplay between signalling pathways contributing to their expression. Here, we have examined the regulation of MMP1 expression, and using in vivo chromatin immunoprecipitation analyses we have demonstrated that cFos bound to the AP-1 cis element within the proximal MMP1 promoter only when the gene was transcriptionally silent as previously observed for MMP13. Subsequent small interfering RNA-mediated silencing confirmed however, that cFos significantly contributes to MMP1 expression. In contrast, silencing of ATF3 (a prime MMP13 modulator) did not affect MMP1 expression whilst silencing of the Wnt-associated regulator cysteine- serine-rich nuclear protein-1 (CSRNP1) resulted in substantial repression of MMP1 but not MMP13. Furthermore, following an early transient peak in expression of CSRNP1 at the mRNA and protein levels similar to that seen for cFOS, CSRNP1 expression subsequently persisted unlike cFOS. Finally, DNA binding assays indicated that the binding of CSRNP1 to the AP-1 consensus-like sequences within the proximal promoter regions of MMP1 and MMP13 was preferentially selective for MMP1 whilst activating transcription factor 3 (ATF3) binding was exclusive to MMP13. These data further extend our understanding of the previously reported differential regulation of these MMP genes, and strongly indicate that although cFos modulates the expression of MMP1/13, downstream factors such as CSRNP1 and ATF3 ultimately serve as transcriptional regulators in the context of an inflammatory stimulus for these potent collagenolytic MMPs.

Protein kinase D3 modulates MMP1 and MMP13 expression in human chondrocytes.

Many catabolic stimuli, including interleukin-1 (IL-1) in combination with oncostatin M (OSM), promote cartilage breakdown via the induction of collagen-degrading collagenases such as matrix metalloproteinase 1 (MMP1) and MMP13 in human articular chondrocytes. Indeed, joint diseases with an inflammatory component are characterised by excessive extracellular matrix (ECM) catabolism. Importantly, protein kinase C (PKC) signalling has a primary role in cytokine-induced MMP1/13 expression, and is known to regulate cellular functions associated with pathologies involving ECM remodelling. At present, substrates downstream of PKC remain undefined. Herein, we show that both IL-1- and OSM-induced phosphorylation of protein kinase D (PKD) in human chondrocytes is strongly associated with signalling via the atypical PKCι isoform. Consequently, inhibiting PKD activation with a pan-PKD inhibitor significantly reduced the expression of MMP1/13. Specific gene silencing of the PKD isoforms revealed that only PKD3 (PRKD3) depletion mirrored the observed MMP repression, indicative of the pharmacological inhibitor specifically affecting only this isoform. PRKD3 silencing was also shown to reduce serine phosphorylation of signal transducer and activator of transcription 3 (STAT3) as well as phosphorylation of all three mitogen-activated protein kinase groups. This altered signalling following PRKD3 silencing led to a significant reduction in the expression of the activator protein-1 (AP-1) genes FOS and JUN, critical for the induction of many MMPs including MMP1/13. Furthermore, the AP-1 factor activating transcription factor 3 (ATF3) was also reduced concomitant with the observed reduction in MMP13 expression. Taken together, we highlight an important role for PKD3 in the pro-inflammatory signalling that promotes cartilage destruction.

Cytokine-induced MMP13 Expression in Human Chondrocytes Is Dependent on Activating Transcription Factor 3 (ATF3) Regulation.

Irreversible breakdown of cartilage extracellular matrix (ECM) by the collagenase matrix metalloproteinase 13 (MMP13) represents a key event in osteoarthritis (OA) progression. Although inflammation is most commonly associated with inflammatory joint diseases, it also occurs in OA and is thus relevant to the prevalent tissue destruction. Here, inflammation generates a cFOS AP-1 early response that indirectly affects MMP13 gene expression. To ascertain a more direct effect on prolonged MMP13 production we examined the potential molecular events occurring between the rapid, transient expression of cFOS and the subsequent MMP13 induction. Importantly, we show MMP13 mRNA expression is mirrored by nascent hnRNA transcription. Employing ChIP assays, cFOS recruitment to the MMP13 promoter occurs at an early stage prior to gene transcription and that recruitment of transcriptional initiation markers also correlated with MMP13 expression. Moreover, protein synthesis inhibition following early FOS expression resulted in a significant decrease in MMP13 expression thus indicating a role for different regulatory factors modulating expression of the gene. Subsequent mRNA transcriptome analyses highlighted several genes induced soon after FOS that could contribute to MMP13 expression. Specific small interfering RNA-mediated silencing highlighted that ATF3 was as highly selective for MMP13 as cFOS. Moreover, ATF3 expression was AP-1(cFOS/cJUN)-dependent and expression levels were maintained after the early transient cFOS response. Furthermore, ATF3 bound the proximal MMP13 AP-1 motif in stimulated chondrocytes at time points that no longer supported binding of FOS Consequently, these findings support roles for both cFOS (indirect) and ATF3 (direct) in effecting MMP13 transcription in human chondrocytes.

Regulation of GTP-binding protein (Gαs) expression in human myometrial cells: a role for tumor necrosis factor in modulating Gαs promoter acetylation by transcriptional complexes.

The onset of parturition is associated with a number of proinflammatory mediators that are themselves regulated by the nuclear factor κB (NF-κB) family of transcription factors. In this context, we previously reported that the RelA NF-κB subunit represses transcription and mRNA expression of the proquiescent Gαs gene in human myometrial cells following stimulation with the proinflammatory cytokine TNF. In the present study, we initially defined the functional consequence of this on myometrial contractility. Here we show that, contrary to our initial expectations, TNF did not induce myometrial contractility but did inhibit the relaxation produced by the histone deacetylase inhibitor trichostatin A, an effect that in turn was abolished by the NF-κB inhibitor N(4)-[2-(4-phenoxyphenyl)ethyl]-4,6-quinazolinediamine. This result suggested a role for TNF in regulating Gαs expression via activating NF-κB and modifying histone acetylation associated with the promoter region of the gene. In this context, we show that the -837 to -618 region of the endogenous Gαs promoter is occupied by cAMP-response element-binding protein (CREB), Egr-1, and Sp1 transcription factors and that CREB-binding protein (CBP) transcriptional complexes form within this region where they induce histone acetylation, resulting in increased Gαs expression. TNF, acting via NF-κB, did not change the levels of CREB, Sp1, or Egr-1 binding to the Gαs promoter, but it induced a significant reduction in the level of CBP. This was associated with increased levels of histone deacetylase-1 and surprisingly an increase in H4K8 acetylation. The latter is discussed herein.

Lysine deacetylase inhibition promotes relaxation of arterial tone and C-terminal acetylation of HSPB6 (Hsp20) in vascular smooth muscle cells.

There is increasing interest in establishing the roles that lysine acetylation of non nuclear proteins may exert in modulating cell function. Lysine deacetylase 8 (KDAC8), for example, has been suggested to interact with α-actin and control the differentiation of smooth muscle cells. However, a direct role of smooth muscle non nuclear protein acetylation in regulating tone is unresolved. We sought to define the actions of two separate KDAC inhibitors on arterial tone and identify filament-interacting protein targets of acetylation and association with KDAC8. Compound 2 (a specific KDAC8 inhibitor) or Trichostatin A (TSA, a broad-spectrum KDAC inhibitor) inhibited rat arterial contractions induced by phenylephrine (PE) or high potassium solution. In contrast to the predominantly nuclear localization of KDAC1 and KDAC2, KDAC8 was positioned in extranuclear areas of native vascular smooth muscle cells. Several filament-associated proteins identified as putative acetylation targets colocalized with KDAC8 by immunoprecipitation (IP): cortactin, α-actin, tropomyosin, HSPB1 (Hsp27) and HSPB6 (Hsp20). Use of anti-acetylated lysine antibodies showed that KDAC inhibition increased acetylation of each protein. A custom-made antibody targeting the C-terminal acetylated lysine of human HSPB6 identified this as a novel target of acetylation that was increased by KDAC inhibition. HSPB6 phosphorylation, a known vasodilatory modification, was concomitantly increased. Interrogation of publicly available mass spectrometry data identified 50 other proteins with an acetylated C-terminal lysine. These novel data, in alliance with other recent studies, alert us to the importance of elucidating the mechanistic links between changes in myofilament-associated protein acetylation, in conjunction with other posttranslational modifications, and the regulation of arterial tone.

A role for cytoskeletal protein acetylation in modulating myometrial activity.

Posttranslational modifications (PTMs) of proteins by phosphorylation are a well-established mechanism by which their activities can be regulated to affect cellular physiology. However, it is becoming increasingly evident that PTMs of proteins by acetylation of lysine residues is also a key effecter in regulating their functional abilities. The best characterized case of this is the epigenetic effects of histone acetyltransferases and deacetylases on gene expression via modulation of nuclear histone acetylation and chromatin remodeling. However, recent published evidence now strongly implicates an important role for nonhistone acetylation in regulating cellular function. In this review, we have considered the potential for regulating myometrial activity not only by epigenetic mechanisms but also by nonepigenetic protein acetylation processes that could directly affect the contractile machinery within these smooth muscle cells.

Expression of the GTP-binding protein Gαs in human myometrial cells is regulated by ubiquitination and protein degradation: involvement of proteasomal inhibition by trichostatin A.

In this study, we show that myometrial transcriptional complexes consisting of Sp1, Sp3, histone deacetylase (HDAC)1/2, RbAp48, and mSin3A are recruited to 4 out of the 6 Sp1-4 sites within the Gαs promoter. Moreover disruption in the binding of these complexes via mithramycin administration results in a substantial decrease in expression of Gαs proteins in myometrial cell cultures. In many instances, these transcriptional regulatory complexes repress expression of genes having a high CG content within their promoter region. This repression can be attenuated by inhibition of HDAC activity by the class I/II HDAC inhibitor trichostatin A (TSA) resulting in increased gene transcription. However, although a substantial increase in Gαs protein levels was observed upon administration of TSA to primary cultures of human myometrial cells, this was not preceded by an increase in messenger RNA (mRNA) and thus an elevation in gene transcription. Importantly the increase in Gαs protein levels occurred via ubiquitination and inhibition of proteasomal activity, indicating that this pathway is also involved in regulating Gαs protein expression during pregnancy and parturition.

Acetylation of heat shock protein 20 (Hsp20) regulates human myometrial activity.

Phosphorylation of heat shock protein 20 (Hsp20) by protein kinase A (PKA) is now recognized as an important regulatory mechanism modulating contractile activity in the human myometrium. Thus agonists that stimulate cyclic AMP production may cause relaxation with resultant beneficial effects on pathologies that affect this tissue such as the onset of premature contractions prior to term. Here we describe for the first time that acetylation of Hsp20 is also a potent post-translational modification that can affect human myometrial activity. We show that histone deacetylase 8 (HDAC8) is a non-nuclear lysine deacetylase (KDAC) that can interact with Hsp20 to affect its acetylation. Importantly, use of a selective linkerless hydroxamic acid HDAC8 inhibitor increases Hsp20 acetylation with no elevation of nuclear-resident histone acetylation nor marked global gene expression changes. These effects are associated with significant inhibition of spontaneous and oxytocin-augmented contractions of ex vivo human myometrial tissue strips. A potential molecular mechanism by which Hsp20 acetylation can affect myometrial activity by liberating cofilin is described and further high-lights the use of specific effectors of KDACs as therapeutic agents in regulating contractility in this smooth muscle.

Epigenetic modulation of the protein kinase A RIIα (PRKAR2A) gene by histone deacetylases 1 and 2 in human smooth muscle cells.

Recently we reported that the expression of the protein kinase A (PKA) regulatory subunit RIIα is dynamically regulated in human smooth muscle cells of the uterus. We showed that expression levels of mRNA/protein were substantially increased during pregnancy and decreased upon labour, changes that were mirrored by particulate type II PKA activity. This implied an important role for RIIα in maintaining uterine quiescence during pregnancy. Consequently the purpose of the present study was to identify potential mechanisms by which expression of the RIIα gene was regulated in this tissue. We indicate here that the three SpI-III (GC) binding domains within the proximal promoter region of the human RIIα gene may play important roles in modulating expression of the gene in human myometrial cells. We show that all three GC binding domains are involved in binding Sp1, Sp3, histone deacetylase (HDACs) 1/2 and RbAp48 transcriptional complexes. The functional significance of these binding domains was further analysed employing in vitro luciferase reporter assays with full-length/truncated RIIα promoter constructs. Importantly we show that treatment of primary human myometrial cell cultures with the general class I/II HDAC inhibitor trichostatin A results in an increase in mRNA/protein levels. Moreover the increase in mRNA levels appeared to be preceded by an increase in aH3, PolIIa, Sp3 and HDAC 2 binding to the three SpI-III (GC) binding sites within the RIIα promoter. These results enable us to provide a model whereby RIIα expression is epigenetically regulated in human myometrial smooth muscle cells by histone deacetylase(s) activity within the GC-rich proximal promoter region of the gene.

Possible dual roles for prostacyclin in human pregnancy and labor.

During pregnancy, the muscular layer of the uterine wall known as the myometrium, which is composed mainly of smooth muscle cells, is maintained in a state of relative quiescence. A switch from myometrial quiescence to myometrial activation is required to establish uterine contractions during labor. Researchers have long been perplexed by the fact that the major prostaglandin produced by the uterus just prior to labor, prostacyclin, is a smooth muscle relaxant. In this issue of the JCI, Fetalvero et al. provide data that they propose explains this paradox, at least in part (see the related article beginning on page 3966). The authors examined uterine tissue from pregnant women near term and found that prostacyclin stimulation, which raises cAMP levels that were previously thought to affect only myometrial quiescence, can promote myometrial activation over time by increasing the expression of a select group of proteins thought to be indicative of a uterine contractile state.

Histone deacetylase inhibitors and a functional potent inhibitory effect on human uterine contractility.

OBJECTIVE: This study was undertaken to investigate the effects of 3 histone deacetylase inhibitors on human uterine contractility. STUDY DESIGN: Biopsy specimens of human myometrium were obtained at elective cesarean section (n = 18). Dissected myometrial strips suspended under isometric conditions, undergoing spontaneous, and oxytocin-induced contractions, were subjected to cumulative additions of 3 histone deacetylase inhibitors: trichostatin A, suberic bishydroxamate (1 nmol/L-10 micromol/L) and valproic acid (100 nmol/L--1 mmol/L). Control experiments were run simultaneously. Integrals of contractile activity were measured by using the PowerLab hardware unit and Chart v3.6 software. Data were analyzed by using 1-way analysis of variance, followed by post hoc analysis. RESULTS: All 3 histone deacetylase inhibitor compounds exerted a potent and cumulative inhibitory effect on spontaneous (n = 18) and oxytocin-induced (n =18) contractility. The mean maximal inhibition values for the 3 compounds were as follows: trichostatin A, 46-54% (P < .05); valproic acid, 35-36% (P < .05); and suberic bishydroxamate, 53-65% (P < .05). CONCLUSION: The histone deacetylase inhibitors trichostatin A, valproic acid, and suberic bishydroxamate exerted a potent inhibitory effect on human uterine contractions. This raises the possibility that this new class of compounds may have tocolytic potential, in addition to their current clinical indications. We speculate that this inhibitory effect may be linked, at least in part, to the ability of histone deacetylase inhibitors to induce the expression of genes involved in maintaining myometrial quiescence via epigenetic mechanisms but may also potentially involve nonepigenetic pathways.

Examining the spatio-temporal expression of mRNA encoding the membrane-bound progesterone receptor-alpha isoform in human cervix and myometrium during pregnancy and labour.

Human parturition is associated with a modification in the sensitivity of the myometrium to progesterone. The molecular basis for this change, however, remains unclear. It is well documented that progesterone can exert its effects through non-genomic mechanisms, including acting through membrane-bound progesterone receptors (mPRs). Recently, a novel membrane-bound PR, termed mPRalpha, was cloned. mPRalpha was unlike any other PR in the databases, but it was seen to have significant homology to G-protein-coupled receptors (GPCR). In this study, we examined the spatio-temporal expression of mPRalpha mRNA in human cervix and both lower and upper myometrial segments from non-pregnant (NP), pregnant (P) and spontaneously labouring (SL) women. We observed an incremental increase in mPRalpha mRNA expression in NP and P samples with the peak level being observed in SL tissues. No major differences were observed between upper or lower pregnant myometrial regions. Interestingly, levels of mPRalpha transcripts were substantially greater in labouring lower segment myometrium compared with labouring upper segment. Significantly, we failed to detect mPRalpha message in either unripe or ripe human cervices. These data suggest that mPRalpha protein function may play a role in regulating lower segment myometrial activity during labour. Whether it functions in the cervix, however, remains unclear.

Novel targeting of cyclooxygenase-2 (COX-2) pre-mRNA using antisense morpholino oligonucleotides directed to the 3' acceptor and 5' donor splice sites of exon 4: suppression of COX-2 activity in human amnion-derived WISH and myometrial cells.

Increased expression of cyclooxygenase-2 (COX-2) has been implicated in the onset of both term and preterm labor. In this context, both selective and nonselective COX-2 inhibitors have been used in clinical trials to determine their efficacy in delaying preterm labor. However, recent evidence indicates that these tocolytics may have potentially adverse fetal and maternal side effects. Therefore, the development of more specific and nontoxic agents to inhibit COX-2 needs to be considered. We have evaluated whether antisense morpholino oligonucleotides have therapeutic potential in inhibiting COX-2 by specifically targeting both the 3' and 5' acceptor and donor sites of exon 4 of COX-2's pre-mRNA sequence. Confocal microscopy on "live" cells illustrated high levels of penetrance of antisense morpholino oligonucleotides using the Endo-Porter formula (Gene-Tools, LLC, Philomath, OR), with delivery efficiencies of 82 and 78%, respectively, in amnion-derived WISH and myometrial cells. Substantial inhibition by the morpholino oligonucleotides of COX-2 expression, induced by lipopolysaccharide administration, was observed at both the mRNA and protein levels. Loss of enzymic activity of COX-2 was confirmed using a sensitive COX enzyme activity assay, which reflects the rate of conversion of arachidonic acid to prostaglandin H2. Our results indicate that antisense morpholino oligonucleotides significantly inhibit expression and activity of this enzyme in in vitro cultures of amnion-WISH and myometrial cells. The potential thus exists that a similar approach can be mimicked in vivo to produce a highly specific and nontoxic strategy to inhibit COX-2 activity with its subsequent effects on the better management of preterm labor and other inflammatory conditions.

Expression of the GTP-binding protein (Galphas) is repressed by the nuclear factor kappaB RelA subunit in human myometrium.

In humans, the factors that govern the switch from myometrial quiescence to coordinated contractions at the initiation of labor are not well defined. The onset of parturition is itself associated with increases in a number of proinflammatory mediators, many of which are regulated by the nuclear factor kappaB (NF-kappaB) family of transcription factors. Recently, we have provided evidence that the RelA NF-kappaB subunit associates with protein kinase A in pregnant myometrial tissue, suggesting links with the Galphas/cAMP/protein kinase A pathway. TNFalpha is a potent activator of NF-kappaB, and levels of this cytokine are increased within the myometrium at term. In the current study, using primary cultures of myometrial cells, TNFalpha was observed to repress expression of Galphas while, at the same time, stimulating NF-kappaB activity. Furthermore, this effect could be replicated by exposure to bacterial lipopolysaccharide and exogenous expression of RelA. Moreover, TNFalpha was seen to repress endogenous Galphas mRNA expression as judged by RT-PCR analyses. Using the chromatin immunoprecipitation assay, we show that RelA did not bind directly to the Galphas promoter. Significantly, expression of a coactivator protein, cAMP response element binding protein binding protein, relieved RelA-induced down-regulation of Galphas expression. Together, these data suggest that, in human myometrium, repression of the Galphas gene by NF-kappaB occurs through a non-DNA binding mechanism involving competition for limiting amounts of cellular coactivator proteins including cAMP response element binding protein binding protein.

The switch in alternative splicing of cyclic AMP-response element modulator protein CREM{tau}2{alpha} (activator) to CREM{alpha} (repressor) in human myometrial cells is mediated by SRp40.

The transcription factor cAMP-response element modulator (CREM) protein, plays a major role in cAMP-responsive gene regulation. Biological consequences resulting from the transcriptional stimuli of CREM are dictated by the expression of multiple protein isoforms generated by extensive alternative splicing of its precursor mRNA. We have previously shown that alternative splicing enables the expression of the CREM gene to be "switched" within the human myometrium during pregnancy from the production of CREMtau(2alpha), a potent transcriptional activator to the synthesis of CREMalpha, a transcriptional repressor. Furthermore we have recently reported that this change in the expression of CREM spliced variants is likely to have important ramifications on the regulation of downstream cAMP-response element-responsive target genes involved in uterine activity during gestation. We have investigated the splicing factors involved in controlling the expression of myometrial CREM splice variants. Data presented here from transient transfections indicate that the switch in the synthesis of CREMtau(2)alpha to CREMalpha that occurs during pregnancy is regulated primarily by an SR protein family member, SRp40. We also show that expression of this splicing factor is tightly regulated in the myometrium during pregnancy. SRp40 regulates the splicing of CREM via its interactions with multiple ESE motifs present in the alternatively exons of CREM. In vitro splicing and electrophoretic mobility shift assays were employed to confirm the functionality of the SRp40-binding ESEs, thus providing a mechanistic explanation of how SRp40 regulates the switch in splicing from production of CREMtau(2)alpha to CREMalpha.

Spatial and temporal expression of the myometrial mitogen-activated protein kinases p38 and ERK1/2 in the human uterus during pregnancy and labor.

OBJECTIVE: We have recently identified a novel putative spliced variant of the activating transcription factor 2 (ATF2) in the human myometrium during pregnancy and labor. This protein, termed ATF2-sm like full-length ATF2, acts as a potent transactivator of cyclic adenosine monophosphate response element (CRE)-containing promoter reporter genes. Similarly, employing microarray gene profiling in myometrial cells, we have shown ATF2-sm to affect the expression of several specific myometrial genes associated with regulating uterine activity during pregnancy and labor. At some point after conception this transcription factor becomes spatially expressed within the body of the uterus, with significantly higher levels detected in the upper (corpus) compared to the lower uterine segment. Because ATF2 species are the primary substrate for phosphorylation by the mitogen-activated protein kinases (MAPKs) p38 and ERK1/2, the purpose of the current investigation was to define the expression levels of these kinases in upper and lower segment myometrium during pregnancy and labor to see if they also correlated with expression of ATF2-sm. METHODS: Paired myometrial samples were collected from the upper (corpus) and lower uterine segments from term nonlaboring and spontaneously laboring women undergoing elective and emergency cesarean deliveries, respectively. Non-pregnant myometrial samples were collected from premenopausal women having hysterectomies for benign gynecologic disorders. The MAPKs p38 and ERK1/2 present in individual myometrial homogenates were resolved using sodium dodecyl sulfate polacrylamide gel electropheresis (SDS-PAGE) with subsequent Western blotting with specific antibodies and scanning densitometry. Expression of the individual MAPKs in myometrial tissues was confirmed in situ using immunohistochemistry. RESULTS: In non-pregnant tissues, p38 and ERK1/2 expression was uniform throughout the uterus. In term pregnant nonlaboring and spontaneously laboring samples expression of p38 and ERK1 was significantly elevated in the upper uterine segment compared to the lower segment, respectively. In contrast, there was no difference in ERK2 expression. CONCLUSION: The data from this study indicate that both p38 and ERK1 are spatially regulated in different uterine regions during pregnancy/labor and suggest that they may be involved in regulating the activity of ATF2 isoforms and their subsequent effects on myometrial function.

Regulation of expression of the chorionic gonadotropin/luteinizing hormone receptor gene in the human myometrium: involvement of specificity protein-1 (Sp1), Sp3, Sp4, Sp-like proteins, and histone deacetylases.

At present there is little information on the regulatory processes by which the chorionic gonadotropin (CG)/LH receptor gene is regulated in the human myometrium during pregnancy and labor. Employing human primary myometrial cell cultures in conjunction with DNA affinity purification assays/Western analysis, DNA binding studies, CG/LH promoter luciferase reporter gene deletion constructs in transfection assays, and measurement of endogenous mRNA levels in vivo by duplex RT-PCR, we have determined the role that the major transcriptional regulatory sequences from the +1 ATG codon to -2678 bp play in modulating expression of the CG/LH receptor gene in the myometrium. We report that the distal -180 to -2678 bp region of the promoter, although capable of binding members of the Jun family via the multiple activator protein-1 sites within this region, has no significant role in regulating the expression of the CG/LH receptor gene in myometrial cells. In contrast, the two specificity protein-1 to -4 (Sp1-4) GC boxes within the +1 to -180 bp proximal promoter are central to expression of the gene in the myometrium. However, not only are Sp1/Sp3 proteins involved in this process, but Sp4 and a novel Sp-like factor(s) also have an intimate part in transcriptional regulation of the gene. It would appear that Sp1/Sp3/Sp4 and Sp-like proteins are involved in recruiting histone deacetylase complexes to the proximal promoter, preventing chromatin remodeling resulting in transcriptional repression of the gene. Our data suggest that administration of the histone deacetylase inhibitor trichostatin A to human myometrial cells in vitro and in vivo substantially removes this silencing effect on expression of the gene and may implicate the use of this and similar agents in increasing myometrial CG/LH receptor levels and subsequent maintenance of uterine relaxation during fetal maturation.