Because multiple myeloma (MM) cells are at risk for endoplasmic reticulum (ER) stress, they require a carefully regulated mechanism to promote protein translation of selected transcripts when proliferation is stimulated. MAPK-interacting kinases (MNKs) may provide this mechanism by enhancing cap-dependent translation of a small number of critical transcripts. We, thus, tested whether MNKs played a role in MM responses to the myeloma growth factor interleukin-6 (IL-6). IL-6 activated MNK1 phosphorylation and induced phosphorylation of its substrate, eIF-4E, in MM lines and primary specimens. MNK paralysis, achieved pharmacologically or by shRNA, prevented MM expansion stimulated by IL-6. A phosphodefective eIF-4E mutant also prevented the IL-6 response, supporting the notion that MNK's role was via phosphorylation of eIF-4E. Both pharmacological MNK inhibition and expression of the phosphodefective eIF-4E mutant inhibited MM growth in mice. Although critical for IL-6-induced expansion, eIF-4E phosphorylation had no significant effect on global translation or Ig expression. Deep sequencing of ribosome-protected mRNAs revealed a repertoire of genes involved in metabolic processes and ER stress modulation whose translation was regulated by eIF-4E phosphorylation. These data indicate MM cells exploit the MNK/eIF-4E pathway for selective mRNA translation without enhancing global translation and risking ER stress. Regulation of protein translation is important in MM because the significant rate of Ig production places the malignant plasma cell at risk for endoplasmic reticulum (ER) stress-induced death. Thus, the MM cell faces a dilemma: although unrestrained translation may be injurious due to enhanced ER stress, the cell requires a regulated mechanism to promote translation when proliferative signals arise. Such a mechanism would allow for the temporary upregulated translation of specific proliferation-dependent proteins but still retain control over global translation. The eIF-4E translation initiation factor could play such a role as it mediates tight regulation of expression of selected proteins The MAP kinase-interacting kinases 1 and 2 (MNK1 and MNK2) initially bind to eIF-4G and subsequently phosphorylate eIF-4E on S209, thus promoting eIF-4E's activity One particular scenario where the MNK kinases and eIF-4E may promote MM tumor growth is during Interleukin-6 (IL-6)-mediated expansion. IL-6-induced MM cell proliferation is mediated by signaling through the ERK MAPK pathway The MM cell lines were obtained from ATCC. The lentiviral vectors targeting MNK1 were purchased from Sigma-Aldrich. The HA-tagged eIF-4E full length coding sequence was isolated from pHA-eIF4E  by PCR. Insert from pLenti6/gfp-lc3 vector was removed by Spe1/Xho1digestion and replaced with Spe/Xho1-digested HA-eIF-4E to yield pLenti6HA-eIF4E. The serine to alanine (SA) and serine to aspartic acid eIF-4E mutants were constructed using the QuikChange site-directed mutagenesis kit (Stratgene). Lentiviral infection of MM cells was performed as previously described Primary cells were purified from marrow of patients as described The MNK kinase assay was carried out as described 7/mouse) were injected subcutaneously (SQ) admixed with matrigel into NOD/SCID mice as described 3, mice were randomized to control (0.5%DMSO), low dose 4-amino-3-(p-fluorophenylamino)pyrazolo pyrimidine (CGP57380) (20 mg/kg) or high dose CGP57380 (50 mg/kg) groups and received daily intraperitoneal (IP) injections. Tumor volume was calculated as previously described 7 EV-transfected 8226 cells in the right flank versus 107 8226 cells transfected with a dominant negative eIF-4E construct. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals in the NIH. The protocol was approved by the IACUC committee of the West LA VA Hospital. All efforts were made to minimize suffering to animals.8226 cells . Results of deep sequencing of total RNA samples and ribosome-protected RNA are posted on the NCBI Sequence Read Archive website with the accession number of SRP033310.Analysis was carried out using the galaxy program. Before alignment, reads were groomed and trimmed to remove artifacts. These reads were first aligned to a database of rRNA sequences to remove potential rRNA contaminating reads. The remaining reads were aligned against a library of transcripts from UCSC known gene database GRCh37/hg19. Comparison of FPKM (fragments per kilobase of exon per million fragments mapped) between ribosome footprints and mRNAs in empty vector or mutant eIF-4E-stably transfected groups were made with Cuffdiff program. Translational efficiency was calculated by FKPM footprint/FKPM mRNA for each transcript. Those with sums of reads from footprint + mRNA above 125 were selected out for further analysis. Comparing translational efficiency identified 166 genes whose translation was significantly inhibited in mutant-expressing cells.Quantitative increases in protein phosphorylation on Western blots were evaluated by densitometric analysis. All Western blots were repeated at least 3 times. The t-test was used to determine significance of differences between groups.Initial experiments tested the ability of IL-6 to activate MNK kinases. MM cell lines were incubated with IL-6 for 3 hrs followed by immunoblot assay for expression of phosphorylated MNK. The antibody used detects phosphorylated MNK1 as well as MNK2. As shown in To ascertain if IL-6 was activating MNK1 or MNK2, we performed in vitro kinase assays . MM cellSubsequent experiments were performed with the ANBL-6 and 8226 MM cell lines as their in vitro growth is known to be affected by exogenous IL-6 o MM cell survival in the absence of IL-6 in both specimens as well as when IL-6 was present.We also had the opportunity to test the effect of the CGP57380 inhibitor against two primary MM cell specimens , cultureAlthough frequently used experimentally as a MNK inhibitor, CGP57380 can inhibit additional kinases as potently as MNK We next used shRNA to knockdown MNK1, MNK2 or both. As shown in To test if MNKs regulate expansion of MM cells via their phosphorylation of eIF-4E, we stably transfected wild type (WT), a serine-to-alanine (SA) mutant (at S209) or serine-to-aspartic acid (ASP) phosphomimetic eIF-4E mutant into ANBL-6 and 8226 MM cells. Although the above described inhibitory effects of our dominant negative eIF-4E mutant strongly support the notion that IL-6-induced, MNK-dependent MM cell growth is, in part, due to a MNK-specific phosphorylation of eIF-4E, MNKs have other substrates that could be playing a role. In particular, MNK kinases can phosphorylate hn RNP A1 (A1) 3, mice were randomized to receive DMSO vehicle alone IP daily, or CGP57380 at 20 mg/kg (low dose (LD)) or 50 mg/kg (high dose (HD)). While MM growth progressed in control mice, both CGP57380 dose cohorts demonstrated an anti-tumor effect which was significant by 4 days of treatment  or the dominant negative eIF-4E mutant (DN). As shown in Mice were also challenged SQ with 10To assess effects on global translation, we utilized the methionine analog azidohomoalanine (AHA) to label newly translated proteins. By using click chemistry, the AHA-incorporating nascent proteins are labeled with a fluorescent dye as described in Since immunoglobulin (Ig) synthesis is a significant portion of protein expression in MM cells, we also tested the effect of the eIF-4E mutant on Ig synthesis. We, thus, tested transfected 8226 cells which synthesize lambda light chains. As shown in 2≤−1.5, false discovery rate <0.05) in mutant-containing MM cells compared to control ANBL-6 cells. A second ribosome profiling experiment gave similar results (correlation coefficient R = 0.941).To identify mRNAs susceptible to translation inhibition due to prevention of eIF-4E phosphorylation, we utilized transcriptome-scale ribosome profiling as previously described 2≤−1.5) but with little effect on mRNA abundance , stearoyl-coA desaturase (SCD) lanosterol synthase (LSS) and mevalonate decarboxylase (MVD). Indeed, interruption of MM cell lipid metabolism is the hypothetical goal of several new therapeutic strategies in myeloma Although several genes specifically involved in proliferation and viability were regulated by eIF-4E phosphorylation, such as cyclin D2 and PIM-2, it was notable that expression of some well known myeloma-associated genes like c-myc and VEGF were not. Immunoblot assay of eIF-4E-mutation-containing MM cells (vs EV cells) confirmed expression of these genes was independent of eIF-4E function (data not shown). One possible explanation for these results is that these genes are translated during IL-6 stimulation through alternative pathways. They have well-defined internal ribosome entry site (IRES) sequences in their 5′UTRs which are highly active in the MM tumor model In summary, our data underscores the importance of MNK kinase activation in the IL-6-induced growth response of MM cells. MNK-induced phosphorylation of eIF-4E upregulates translation of a select number of transcripts, which are important for the response, but does not significantly increase global translation. As the MNKs are dispensable for normal physiology Figure S1ANBL-6 MM cells cultured +/− CGP (25 uM) for 72 hrs in the presence or absence of increasing concentrations of rIL-6. Data represent number of viable cells recovered, mean+/−SD, n = 3. CGP significantly (p<0.05) reduced the number of viable cells at all concentrations of IL-6 (but not in the absence of IL-6).(TIF)Click here for additional data file.Figure S2ANBL-6 or 8226 cells cultured +/− IL-6 (500 U/ml) and +/− CGP (25 uM) for 48 or 72 hrs after which apoptosis assayed by flow cytometric analysis of activated caspase 3. Data are means of 3 separate experiments. SDs were <10% of the means for all groups (not shown). As a positive control, staurosporine, at 1 uM, induced ANBL-6 and 8226 apoptosis .(DOC)Click here for additional data file.Figure S3A) shRNA-transfected 8226 MM cells to silence hn RNP A1 showing loss of A1 expression vs EV cells (transfected with shRNA expressing a scramble sequence (SCR)). B) MM cells expressing empty vectors (EVs) or shRNA for hn RNP A1 (sh1 or sh2) or wild type (WT) eIF-4E or SA dominant negative eIF-4E phospho-mutant. Cells stimulated +/− LPS and TNF-alpha expression assessed by ELISA. Data are means+/−SD of 3 experiments. *denotes statistically significant difference (p<0.05) from corresponding EV cells.(TIF)Click here for additional data file.Figure S4Proliferation, RNA-binding protein, and translational machinery genes showing inhibited translational efficiency due to eIF-4E mutation.(TIF)Click here for additional data file.Figure S5Cytoskeleton, adhesion proteins & other genes that demonstrate inhibited translational efficiency due to eIF-4E mutation.(TIF)Click here for additional data file.


TGR5  is a plasma membrane-bound, G protein-coupled receptor for bile acids, expressed in many human cells. The aim of this study was to describe that targeting TGR5 could provide an exciting new pharmacological approach to improve different aspects of the metabolic syndrome in humans.The data on pharmacological targeting of TGR5 have been provided from more than eighty review and original scientific articles, published from 2007 to 2012. The research was performed using the following key words: bile acids, TGR5, metabolism, diabetes, obesity.A dietary supplementation of bile acids (BAs) significantly reduced body weight in mice fed with a fat-rich diet. It was the consequence of the induction of deiodinase 2 (D2) through a TGR5/cAMP-mediated pathway. D2 is able to induce the conversion of inactive thyroxine (T4) into the active 3,5,3′-tri-iodothyronine (T3), which enhances the energy expenditure in brown adipose tissue (BAT) and skeletal muscle myoblasts. TGR5 induces glucagon-like peptide-1 (GLP-1) secretion in cultured mouse enteroendocrine STC-1 cells. This property contributes to beneficial effects of TGR5 on glucose metabolism and improves insulin sensitivity. TGR5 activation in mice decreased serum and liver triglyceride levels. The anti-inflammatory action of TGR5 in mouse macrophages attenuated the development of atherosclerotic lesions and could contribute to protective effects of TGR5 on liver steatosis.TGR5 may be targeted by natural compounds as well as by synthetic agonists. Despite the fact that targeting TGR5 in animals brings great promise for metabolic syndrome treatment, multiple studies described the side effects of targeting TGR5 and further clinical studies are needed to evaluate and identify safe and efficient TGR5 agonists.


We demonstrate that a terminal splice acceptor site is essential to establish coupling with the poly(A) signal in a chromosomally integrated β-globin gene. This is in part to alleviate the suppression of 3′ end formation by U1 small nuclear RNA, which is known to bind pre-mRNA at the earliest stage of spliceosome assembly. Interestingly, blocks to splicing that are subsequent to terminal splice acceptor site function, but before catalysis, have little observable effect on 3′ end formation. These data suggest that early stages of spliceosome assembly are sufficient to functionally couple splicing and 3′ end formation, but that on-going intron removal is less critical.Splicing of human pre-mRNA is reciprocally coupled to 3′ end formation by terminal exon definition, which occurs co-transcriptionally. It is required for the final maturation of most human pre-mRNAs and is therefore important to understand. We have used several strategies to block splicing at specific stages  First, U1 snRNA (U1) contacts the 5′ splice site after which U2 snRNA (U2) is recruited to the branch-point following recognition of the 3′ splice site and polypyrimidine tract by U2AF35 and 65, respectively. U4, U5 and U6 snRNAs then join, and two-step catalysis occurs after U1 and U4 are ejected from the spliceosome  (pA) signal within the RNA, which is composed of a hexameric sequence, usually AAUAAA, followed by a U or G/U rich sequence ,5. CleavFirst observed 30 years ago , it is n3′ end formation is also negatively regulated by certain splicing factors. Early experiments showed that 5′ splice site sequences are involved in silencing some viral pA signals through the recruitment of U1 snRNA . More rein vivo  and by inhibiting U4 snRNA. These approaches reduced splicing but surprisingly had little effect on 3′ end formation. Thus, a tSA site establishes coupling between splicing and 3′ end formation, which is sufficient for cleavage and polyadenylation even when intron removal is inefficient.The β-globin plasmid for Cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% foetal calf serum. SSA was used at 50 ng/ml unless otherwise stated. Actinomycin D (ActD) was used at 10 μg/ml. Electroporation was performed on a confluent 10-cm diameter dish of cells using 10–15 μM anti-sense morpholino (AMO) in 400 μl of Dulbecco’s modified Eagle’s medium using a 4-mm gap cuvette . RNA was isolated 3 h after electroporation. For transient transfection, 2.5 μg of reporter and 0.5 μg VA were introduced using Xtreme gene (Roche). For anti-sense oligonucleotide (ASO) experiments, 500 pmol of oligo was introduced into a 24-well dish of cells using Lipofectamine RNAiMax (Life Technologies). For all plasmid and ASO transfections, RNA was isolated after 24 h. For stable integration, a 6-cm dish of Flp-IN HEK cells was transfected with 1 μg of the appropriate β-globin plasmid in addition to 3 μg of pOG44 using JetPrime (Polyplus) transfection reagent. Forty-eight h post-transfection, cells were transferred to a tissue culture flask and grown in the presence of Hygromycin (100 μg/ml) and Blasticidin (10 μg/ml) until resistant cells emerged. Transcription was induced by overnight growth in media supplemented with 1 μg/ml tetracycline (tet).Pol II ; anti-CstF77 ; anti-U2AF65 .Chromatin-associated and nucleoplasmic RNA was isolated as previously described ,33. RNA supplementary material.Detailed protocols are provided in the supplementary material.Primer, AMO and ASO sequences are provided in the To analyse coupling between splicing and 3′ end formation in a chromosomal context, we constructed two HEK cell lines containing either a single stably integrated wild-type (βWT) β-globin gene or one with a mutated tSA site (βtSA) A. TranscSupplementary Figure S1).To test whether the βtSA mutation affected 3′ end formation in the chromosomal context as it does in plasmids , we perfAs pre-mRNA processing defects often reduce transcription , we testMutation of a tSA site is also predicted to prevent the recruitment of relevant 3′ splice site-binding factors that participate in terminal exon definition. We tested this by performing RNA immunoprecipitation (RIP) to analyse binding of the 65 kDa component of U2AF, U2AF65, to the tSA site of βWT or βtSA transcripts. Immunoprecipitated RNA was reverse transcribed with random hexamers and then PCR amplified with primers across the tSA site. Histone H1E mRNA was used as a negative control, as U2AF65 is not expected to bind this transcript. A 4–5-fold greater proportion of βWT tSA site-containing transcripts were recovered in comparison G. In conIn addition to blocking the recruitment of factors that may act positively in 3′ end formation, a tSA site mutation may also leave the pA signal vulnerable to negative effects. In particular, U1 inhibits cleavage and polyadenylation at pA sites when bound to nearby 5′ splice sites ,21,22,42To determine the effect of U1 inhibition on β-globin splicing, reverse transcription and real-time PCR were used to quantitate the level of unspliced intron 2 (Ex2-In2), intron 2 lariat, spliced (Ex2-Ex3) and total (Ex3) β-globin transcripts B and C. Supplementary Figure S2). U1 inhibition caused a 2-fold increase of this species in βWT cells, suggesting that 3′ end formation occurs when U1 is not recruited to the natural intron. Interestingly, a similar effect on 3′ end processing was observed in βtSA cells. Furthermore, an AMO targeting U1 gave the same result in βtSA plasmid transfected HeLa cells treated for only 3 hours . Little SSA-dependent change in total or spliced βWT RNA was seen potentially because stable processed transcripts are present from before this short splicing block. As expected, total and correctly spliced βtSA transcripts were present at low levels or absent, respectively.We first determined the effect of SSA on β-globin splicing using both the βWT and βtSA HEK cell lines. Total RNA was isolated from cells treated with SSA or its methanol (MeOH) solvent. Treatment was limited to 3 h to limit potential off-target effects of splicing inhibition. Real-time PCR was then used to quantitate the level of unspliced intron 2 (Ex2-In2), intron 2 lariat, spliced (Ex2-Ex3) and total (Ex3) β-globin transcripts A and B. To analyse 3′ end formation, we detected β-globin transcripts that were not cleaved at the pA site C. We alsTo further determine the effect of SSA on β-globin 3′ end formation, we used another cell line (βWTCoTC). This is the same as βWT but has a sequence (the CoTC element) in the 3′ flank, which promotes transcriptional termination in both plasmid and chromosomal environments ,34. An eIf 3′ end formation does continue in the presence of SSA, cleaved and polyadenylated transcripts that are also unspliced will accumulate. To test this and any function for a tSA site, we used our 3′ RACE approach to detect these transcripts E. SSA inWe next wanted to analyse the effects of U1 inhibition and SSA on transcripts synthesised from genes in their natural chromosomal context. P27 and Myc transcripts were chosen, as they have previously been used to study effects of SSA ,49. MoreSupplementary Figure S4). This is again consistent with our β-globin analyses.We next analysed the effects of blocking later steps in spliceosome assembly using SSA. Total RNA was isolated from cells treated with MeOH or SSA. Following reverse transcription, cDNA was analysed with primers to detect unspliced, non-pA cleaved and spliced RNA from Myc and P27 genes C. In botXenopus Oocytes that contain U4 lacking this region can only associate with U1 and U2 .We found it surprising that SSA-induced splicing inhibition did not observably impact on 3′ end formation. To be more confident about this finding, we sought an alternative means of inhibiting a similar step of splicing and used an AMO directed to U4 A. The re1 and U2 . Consistin vivo by electroporating it into HeLa cells that were transiently transfected with a β-globin expression plasmid .We validated the effectiveness of the U4 AMO  plasmid B. Total Supplementary Figure S6B and C). Analysis of prematurely processed NR3C1 revealed that the ability of U1 to bind pre-mRNA and inhibit premature 3′ end formation is largely unaffected by U4 AMO treatment .We next tested whether the reduced β-globin splicing caused corresponding effects on 3′ end formation. We performed S1 nuclease analysis on the same RNA samples as It is well established that there is an intimate relationship between pA signal function and transcriptional termination. Therefore, as a final measure of the effect of U4 inhibition on 3′ end formation, we checked its effect on transcriptional termination D. RNA waSupplementary Figure S8). A lack of effect on processing at the P27 and Myc pA sites was also confirmed using an anchored oligo-dT reverse transcription and real-time PCR approach .We next analysed the effect of U4 inhibition on endogenous P27 and Myc transcripts by performing qRT-PCR to detect unspliced, spliced and non-pA cleaved sampled in control or U4 AMO-treated cells A. We obsSupplementary Figure S7). This is consistent with the prediction, from previous data and from the classical splicing pathway . More generally, most splicing factors described as having potential function in cleavage and polyadenylation are associated with either U1 or U2 recruitment (U2AF65 is involved in 3′ splice site recognition, terminal exon definition and has been shown to stimulate cleavage and polyadenylation. Interestingly, U2AF65 can promote β-globin 3′ end processing when tethered artificially to the transcript, suggesting that this function does not require removal of an intron . Our finin vitro . SF3b isruitment ,40,58. TGlobal analyses of nascent transcripts show co-transcriptional removal of a large number of introns ,26. HoweIt is well documented that many splice defective transcripts in lower and higher eukaryotes are rapidly degraded ,53,63. AWe have shown that the tight co-transcriptional coupling between splicing and 3′ end formation is established early during terminal exon definition following U1 recruitment to the 5′ splice site and recognition of the tSA site. These early events seem sufficient to permit 3′ end formation, which continues following blocks to later steps in the splicing process.Supplementary Data are available at NAR Online: Supplementary Table 1, Supplementary Figures 1–10 and Supplementary Methods.Wellcome Trust [088499/Z/09/2] and Centre core grant from the Wellcome Trust [092076]. Funding for open access charge: The Wellcome Trust.Research career development award from the Conflict of interest statement. None declared.


Plocamium cartilagineum is a common red alga on the benthos of Antarctica and can be a dominant understory species along the western Antarctic Peninsula. Algae from this region have been studied chemically, and like “P. cartilagineum” from other worldwide locations where it is common, it is rich in halogenated monoterpenes, some of which have been implicated as feeding deterrents toward sympatric algal predators. Secondary metabolites are highly variable in this alga, both qualitatively and quantitatively, leading us to probe individual plants to track the possible link of variability to genetic or other factors. Using cox1 and rbcL gene sequencing, we find that the Antarctic alga divides into two closely related phylogroups, but not species, each of which is further divided into one of five chemogroups. The chemogroups themselves, defined on the basis of Bray-Curtis similarity profiling of GC/QqQ chromatographic analyses, are largely site specific within a 10 km2 area. Thus, on the limited geographical range of this analysis, P. cartilagineum displays only modest genetic radiation, but its secondary metabolome was found to have experienced more extensive radiation. Such metabogenomic divergence demonstrated on the larger geographical scale of the Antarctic Peninsula, or perhaps even continent-wide, may contribute to the discovery of cryptic speciation. Plocamium cartilagineum is widely reported in the world’s oceans, including the cold waters of the Arctic and Antarctic, temperate waters of Europe, Asia, Africa and the Americas, and tropical waters of the Pacific, Atlantic and Indian Oceans [P. cartilagineum are polyhalogenated monoterpenes, and many of them bear pharmacological properties, such as cytotoxicity or antibiotic activity [The rhodophyte n Oceans . Systeman Oceans . Indepenn Oceans . More thactivity .Plocamium cartilagineum has a circumpolar distribution [. The systematics and taxonomy of “P. cartilagineum” is in flux in Antarctica, as elsewhere [P. coccineum, which is now considered a synonym of P. cartilagineum [P. hookeri and P. secundatum have also been reported from some locations along the western Antarctic Peninsula (WAP) [P. cartilagineum, but their distinctiveness illustrates that there is notable morphological variability within Antarctic P. cartilagineum [In Antarctica, ribution ,6, altholsewhere . Early wlsewhere ,7,8,9 relagineum . The Sublagineum .P. cartilagineum from shallow water habitats, where it can be locally dominant. Bulk collections of algae, comprising mixed populations from numerous collecting sites, produced extracts that were used in assays to ascertain levels of deterrence toward potential predators [Gondogeneia antarctica, though lack of sufficient material hindered screening of the compounds against potential fish predators [P. cartilagineum serves as host to several amphipod species, where they gain protection from fish predation, due to the physical structure of the algal thallus and feeding deterrent effects from metabolites found in its extracts [P. cartilagineum, inhibiting overgrowth that might otherwise limit the photosynthetic potential of the alga [In recent field seasons at Palmer Station , on the WAP, we have collected redators . Field sredators . P. cartextracts . Most ofextracts , but in the alga .P. cartilagineum complicated tissue- and extract-based assays used in evaluating the extent to which these compounds protected the alga from predation, prompting us to profile individual plants. We report here a detailed metabogenomic analysis of site specificity and distribution of halogenated monoterpenes in two phylogroups of P. cartilagineum from the western Antarctic Peninsula.Collection-to-collection variability observed in the suite of secondary metabolites from WAP Plocamium cartilagineum that combine plant material collected from multiple sites within the small boating range around Palmer Station, Antarctica, display a breadth of secondary metabolites  extracts of the three members of chemogroup 4, each individual of which  mass spectrometry, a technique especially well-suited to halogenated compounds , showed  site 5, . Six proic acids .Relative intensities of the chromatographic features observed in P. cartilagineum, we chose to sequence the rbcL and cox1 genes to examine genetic diversity within our collection [cox1 genes from our algal samples diverged into two clades , GC/MS data was analyzed statistically. The same five chemogroups distinguish themselves from one another at approximately 50% Bray-Curtis similarity . Chemogr2  and chemogroup . Algal collection sites were within 3 km of Palmer Station, an area encompassing approximately 10 kmPortieria hornemannii, encompassing only a slightly larger geographical range, found no reproducible site specificity among secondary metabolites [P. hornemannii found that light availability, which could potentially vary from site-to-site, might influence the quantitation of a halogenated monoterpene [P. cartilagineum have been studied [Such site specificity is surprising. Similar studies of the red alga abolites ,23. Howeoterpene . The eff studied , and it  studied ,26. PAR,P. cartilagineum at multiple sites and depths in this study area. Although counts of individuals have not been made, the fauna is qualitatively similar across all collections . However, there do appear to be quantitative differences across sites in the numbers of at least some specific amphipod species . The possibility that these differences are either causes or consequences of the chemical diversity remains to be explored.Another factor that may vary across both depth and site is predation pressure. By far the most abundant grazers in the community are amphipods ,28,29. ALight and wave energy also vary with exposure, with more exposed sites generally having clearer, open ocean water more frequently. Greater distance from glaciers (and their silty melt water) also generally corresponds with clearer waters. However, no consistent patterns emerge when considering these factors, for example, algae from the most exposed site, Norsel Point wall (site 2) group with two individuals from a moderately protected site at Hermit Island (site 1). The Hermit site has less glacial input, so clearer waters than some of the other protected sites, but not the other Hermit individual (1B) groups with two individuals from Hero Inlet, which is by far the most protected site with the most glacial influence and most turbid water. The other Hero Inlet individual (6C), groups with individuals from Old Palmer, which is probably the second most protected and second most glacial-influenced site, although in both cases, much less so than Hero Inlet.Bahia Paraiso shipwreck  where a single site had multiple chemogroups, the algae were also in different phylogroups. However, it is possible that some of the within-chemogroup diversity in secondary metabolites could be related to algal life history stage.We are aware of two reports of secondary metabolite variations in red algae that are related to life history stage ,30, inclP. cartilagineum was hand collected by scuba diving between February and April, 2010, from multiple dive sites around Palmer Station on Anvers Island, Antarctica . Periodic collections were cleaned of foreign materials in the Palmer Station aquarium, then frozen at −20 °C, with subsequent collections combined to produce this mixed population that was used for chemical isolation and characterization studies at our home institutions. Individual thalli used in this study were collected in late February 2012 from seven dive sites in the vicinity of Palmer Station , epi-plocamene D (2), and -1,8-dibromo-3,4,7-trichloro-3,7-dimethylocta-1,5-diene (3) were characterized by comparison of their 1H and 13C NMR spectra to that reported in the literature [1 and 3 were also subject to X-ray crystallographic analysis.Mixed population algae from the 2010 collection were thawed, then extracted in 3:1 dichloromethane/methanol. The concentrated lipophilic extract was examined by GC/MSm as described below, then purified by repeated medium pressure silica gel chromatography (100% hexane to 100% ethyl acetate on 4 g or 12 g Teledyne/Isco silica gel cartridges)m followed by repeated high pressure liquid chromatography . Anverene , concentrated under a stream of nitrogen gas, then transferred to a GC/MS sample vial in methanol to a final concentration of 1 mg/mL. Extracts were analyzed on an Agilent 7980A GC interfaced to an Agilent 7000 series QqQ mass spectrometer operating in either electron ionization (EI) or chemical ionization (CI) mode, the latter using methane at 2 mL/min as the ionization gas. Injections of 1 μL of the algal extract solution were vaporized on the preheated splitless inlet at 250 °C, then introduced onto an HP-5ms column (30 m × 0.25 mm i.d.) using a 35 min temperature gradient . Helium was used as a carrier gas at a constant flow rate of 1 mL/min.cox1 mitochondrial gene [rbcL gene [cox1 amplification began with an initial denaturation at 95 °C for 2 min, followed by 5 cycles of 30 s at 95 °C, 30 s at 45 °C and 1 min at 72 °C; then 35 cycles of 30 s at 95 °C, 30 s at 46.5 °C and 1 min at 72 °C; and finally, a 7 min extension at 72 °C. rbcL amplification initiated with a 3 min denaturation at 94 °C, then 40 cycles of 90 s at 94 °C, 2 min at 37 °C, 3 min at 72 °C, terminating with 4 min extension at 72 °C. Duplicate PCR products were pooled and purified with Promega Wizard prior to sequencing on an ABI PRISM 3730. Six-hundred sixty base pairs were read for cox1 and 890 bp for rbcL. Sequences were aligned by eye and with the software Geneious  using the MUSCLE plugin [cox1 and rbcL. Maximum likelihood phylogenetic analysis was performed on the CIPRES Science Gateway [cox1 A and B and rbcL A and B are KF158990, KF158991, KF158993 and KF158992 respectively.DNA was extracted from silica dried tissue (~10 mg) using the Qiagen DNeasy Plant mini kit. Specific primers amplified the ial gene  and the bcL gene . PCR ampE plugin . AlignmeE plugin ,35. The  Gateway  using GAThe GC/MS data was analyzed in Agilent’s MassHunter Qualitative B5.01, where 43 recurring compounds were identified between 10 and 30 min throughout the 21 samples. The height of these peaks relative (>10%) to that of the most abundant peak within each chromatogram was calculated and input into PRIMER-6 for statistical analysis. The peak height data for all peaks >10% of the largest peak were analyzed for resemblance using Bray-Curtis similarity and clustered using the group average.Plocamiumcartilagineum collected from the vicinity of Palmer Station, Antarctica, diverges both chemically and genetically, but should still be considered as a single species. We see site-specific differences in both phylogeny and secondary metabolism between closely spaced sites  and also similarities between more distantly related sites . Observable environmental factors do not appear to influence site specificity. P. cartilagineum on the WAP may be undergoing cryptic speciation as is seen elsewhere, and its chemical diversity could be either a partial consequence or even a partial driver of that, but a broader view that incorporates similar data from algae more distant on the WAP, or from other sites on the continent, needs to be considered.


However, several sites of tyrosine autophosphorylation have recently been identified establishing that BRI1 is a dual-specificity kinase. This raises the paradox that BRI1 contains phosphotyrosine but was only observed to autophosphorylate on serine and threonine sites. In the present study, we demonstrate that autophosphorylation on threonine and tyrosine (and presumably serine) residues is a post-translational modification, ruling out a co-translational mechanism that could explain the paradox. Moreover, we show that in general, autophosphorylation of the recombinant protein appears to be hierarchical and proceeds in the order: phosphoserine > phosphothreonine > phosphotyrosine. This may explain why tyrosine autophosphorylation was not observed in some studies. Finally, we also show that the juxtamembrane domain of BRI1 is an activator of the kinase domain, and that kinase specificity (serine/threonine versus tyrosine) can be affected by residues outside of the kinase domain. This may have implications for identification of signature motifs that distinguish serine/threonine kinases from dual-specificity kinases.In metazoans, receptor kinases control many essential processes related to growth and development and response to the environment. The receptor kinases in plants and animals are structurally similar but evolutionarily distinct and thus while most animal receptor kinases are tyrosine kinases the plant receptor kinases are classified as serine/threonine kinases. One of the best studied plant receptor kinases is Brassinosteroid Insensitive 1 (BRI1), which functions in brassinosteroid signaling. Consistent with its classification, BRI1 was shown in early studies to autophosphorylate  Arabidopsis contains more than 600 receptor-like kinases that are structurally and functionally similar to animal receptor kinases but are evolutionarily distinct. Animal receptor kinases are predominantly tyrosine kinases while plant receptor kinases are generally classified as serine/threonine kinases. However, recent work suggests that at least some plant receptor kinases are dual-specificity kinases that can autophosphorylate on serine, threonine, and tyrosine residues  emerges as an additional component that may contribute to BR signaling in planta and supports the recent report of BRI1-mediated phosphorylation of BKI1 at Tyr-211  the activating role of the juxtamembrane domain; and (2) the paradox of tyrosine autophosphorylation. With respect to the juxtamembrane domain, we showed in a previous study , SRF9 , FLS2 , At2g23950 (residues 259–634), BRI1 , BAK1 , BKK1 , BRL1 , or BRL3  were cloned into a Gateway modified pFlag-Mac vector and expressed in BL21 (DE3) pLysS cells . As specified, serial truncations of the juxtamembrane domain of BRI1 CD were made by sequentially removing five amino acid segments between residue 815 and 875. The truncation mutants were cloned and expressed as for the intact CDs. The recombinant Flag-tagged proteins were immunopurified using anti-Flag M2 affinity gel . After elution from the beads, the protein solutions were dialyzed against a 1000× volume of buffer containing 20 mM MOPS, pH 7.5, and 1 mM DTT as previously described  with bovine serum albumin as the standard. Proteins were separated on 12% polyacrylamide (0.1% SDS) gels and transferred to polyvinylidene difluoride (PVDF) fluorescence-specific membranes . Membranes were blocked in a 2% (v/v) fish gelatin solution in phosphate-buffered saline  before being incubated with primary antibodies, which were diluted as specified in PBS containing 0.1% (v/v) Tween-20 (PBST). Blots were probed with custom polyclonal antibodies that recognize BRI1 phosphotyrosine-831 , phosphotyrosine-956 , or phosphotyrosine-1072 . In general, experiments were performed at least twice and representative results are presented.Recombinant protein preparations were mixed with four-volumes of pre-heated (95°C) 1× SDS-PAGE sample buffer containing 1 M urea, 0.7 M 2-mercaptoethanol, 5 mM NaF, 1 mM NaE. coli cells expressing Flag-BRI1 CD were harvested before addition of IPTG or 16 h after addition of IPTG to induce recombinant protein production. Bacterial cells were lysed and debris was removed by low speed centrifugation. The majority of the Flag-BRI1 protein was removed by absorption using anti-Flag M2 affinity gel  and the remaining proteins were processed for 2-DE. Total E. coli proteins were precipitated with five volumes of ice-cold 0.1 M ammonium acetate in methanol and left at −20°C overnight to precipitate total soluble proteins. After centrifugation at 24,000 g for 20 min, the protein pellet was washed three times with 1 ml with ice-cold 0.1 M ammonium acetate in methanol, and once with ethanol before resuspension in 300 μl IEF sample loading buffer  with vortexing. The resuspending pellet was then incubated at 30°C for 1 h, centrifuged at 24,000 g for 20 min, and the resulting supernatant was transferred to a new tube. The protein concentration was determined with Bio-Rad protein assay using BSA as a standard and the 2-DE analysis was performed as described . Reactions were incubated at room temperature and peptide kinase activity was measured using the filter paper binding assay. Values reported are means ± SEM and are representative of at least two independent experiments.Peptide substrate phosphorylation assays were performed as described  for 16 h and the tryptic peptides were extracted with 0.5% TFA and 50% acetonitrile and dried. The collected peptides were further purified with C18 Ziptips , before analysis in the Voyager DE-STR MALDI-TOF . The matrix was 1% phosphoric acid in 2,5-dihydroxybenzoic acid  directed mutant, referred to as mBRI1, that is completely unphosphorylated  that inhibit peptide kinase activity, and of the sites monitored, Ser-891  confirming that they were not cross-linked or dimeric forms of Flag-BRI1, but rather represent transphosphorylation of E. coli proteins catalyzed by Flag-BRI1.We recently reported that g Figure A and crog Figure B and antg Figure D antibods Figure D. ProQ Ds Figure , but onls Figure C, and ths Figure B. The liE. coli proteins by recombinant BRI1, total soluble proteins from bacterial cells at time zero, or after 16 h of induction were resolved by 2-DE and blots were probed with anti-phosphotyrosine antibodies . The basis for the lack of autophosphorylation of the RD-type kinase, At2g23950, could indicate that the recombinant protein did not fold properly, that phosphorylation of activation loop residues is catalyzed by another protein kinase, or simply that autophosphorylation does not occur with this particular kinase. For the three proteins that did not autophosphorylate (by ProQ Diamond staining), immunoblotting with anti-Flag antibodies cross-reacted with single bands although in each case Coommassie staining detected minor contaminating proteins that were not related to the recombinant protein kinase.We also tested the CDs of several other leucine-rich repeat receptor-like kinases (LRR-RLKs) for their ability to transphosphorylate s Figure A. The RDs Figure . As showE. coli proteins and a variable amount of residual recombinant protein) were analyzed for protein phosphorylation to identify which receptor kinases could catalyze transphosphorylation of E. coli proteins. Of those tested, BRI1 could strongly transphosphorylate on threonine and tyrosine residues as expected, while BRL1 and BRL2 could only transphosphorylate E. coli proteins weakly on threonine residues and not at all on tyrosine. Because transphosphorylation of E. coli proteins by BRL1 and BRL3 onthreonine residues was markedly reduced relative to BRI1, but total transphosphorylation as monitored by ProQ Diamond staining was similar among the three receptor kinases, it would appear that BRL1 and BRL3 are primarily transphosphorylating E. coli proteins on serine residues. Thus, it appears that the three receptor kinases may differ in specificity of transphosphorylation (serine versus threonine versus tyrosine). It is also worth noting that inability of some receptor kinases  to transphosphorylate E. coli proteins cannot be attributed to inherent lack of catalytic activity, and therefore autophosphorylation and transphosphorylation activities are not directly related to one another.Following purification of the recombinant CDs, the remaining soluble-protein supernatants  is hierarchical; (ii) is post-translational rather than co-translational; (iii) likely involves both intra- and inter-molecular events; and (iv) is activated by the juxtamembrane domain. The hierarchical nature of autophosphorylation was evident during production of recombinant Flag-BRI1 in e Figure A, althoue Figure B. Exceptin vitro autophosphorylation, only 32P-labeling of serine and threonine residues was observed, and thus emerged the paradox of tyrosine autophosphorylation. One resolution to the paradox would be that autophosphorylation on tyrosine residues at least was a co-translational process, as observed for the GSK3 and DRKY family protein kinases. Several lines of evidence suggest that autophosphorylation of Flag-BRI1 protein on serine, threonine, and tyrosine residues occurs as a result of a traditional post-translational modification. First, if autophosphorylation was co-translational there would be no increase in autophosphorylation during induction; i.e., autophosphorylation normalized for BRI1 protein would be constant and that is not the case , we reported earlier  mutant, suggesting an inter-molecular reaction . This suggests that intrinsic tyrosine phosphorylation activity may be much more restricted or specific than serine/threonine phosphorylation; i.e., fewer tyrosine phosphorylation motifs are recognized. Alternatively, it may also reflect in part the fact that serine and threonine are together about fourfold more abundant in proteins compared to tyrosine. In the complete Expasy proteome database (www.expasy.org), the frequencies of serine, threonine, and tyrosine are 6.6, 5.3, and 2.9%, respectively. Insights learned from the bacterial system may help to answer these questions and thereby facilitate identification of novel plant proteins that are substrates of BRI1 in vivo.The transphosphorylation of 1 Figure  emerges  Figures B and D. in situ (during expression of Flag-BRI1 in E. coli) and the mechanism established as a post-translational modification. Had the mechanism involved a co-translational modification, tyrosine phosphorylation would not have been a readily reversible modification and given that BRI1 is a relatively long-lived receptor kinase .Tyrosine autophosphorylation of recombinant Flag-BRI1 CD is shown to occur via a post-translational modification, as opposed to a co-translational modification, and the ability of BRI1 to transphosphorylate proteins on tyrosine residues (in addition to serine and threonine) is documented. It is interesting that transphosphorylation of The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ucp1, lipolysis, cAMP levels, and phosphorylation of hormone sensitive lipase in response to isoproterenol or forskolin. Noncanonical IKKs reduce catecholamine sensitivity by phosphorylating and activating the major adipocyte phosphodiesterase PDE3B. In vivo inhibition of these kinases by treatment of obese mice with the drug amlexanox reversed obesity-induced catecholamine resistance, and restored PKA signaling in response to injection of a β-3 adrenergic agonist. These studies suggest that by reducing production of cAMP in adipocytes, IKKε and TBK1 may contribute to the repression of energy expenditure during obesity.Obesity produces a chronic inflammatory state involving the NFκB pathway, resulting in persistent elevation of the noncanonical IκB kinases IKKε and TBK1. In this study, we report that these kinases attenuate β-adrenergic signaling in white adipose tissue. Treatment of 3T3-L1 adipocytes with specific inhibitors of these kinases restored β-adrenergic signaling and lipolysis attenuated by TNFα and Poly (I:C). Conversely, overexpression of the kinases reduced induction of DOI:http://dx.doi.org/10.7554/eLife.01119.001 Obesity is a complex metabolic disorder that is caused by increased food intake and decreased expenditure of energy. Obesity also increases the risk of developing type 2 diabetes, heart disease, stroke, arthritis, and certain cancers. There is considerable evidence to suggest that adipose tissue becomes less sensitive to catecholamines such as adrenaline in states of obesity, and that this reduced sensitivity in turn reduces energy expenditure. However, the details of this process are not fully understood.It is well established that obesity generates a state of chronic, low-grade inflammation in liver and adipose tissue, accompanied by the secretion of signaling proteins that prevent fat cells from responding to insulin, which leads to type 2 diabetes. Activation of the NFκB pathway is thought to have a central role in causing this inflammation. Now Mowers et al. have investigated whether inflammation caused by activation of the NFκB pathway also has a role in producing catecholamine resistance in fat cells.Obesity-dependent activation of the NFκB pathway increases the levels of a pair of enzymes, IKKε and TBK1. Mowers et al. found that elevated levels of these two enzymes reduced the ability of certain receptors  in the fat cells of obese mice to respond to catecholamines. High levels of the two enzymes also resulted in lower levels of a second messenger molecule called cAMP, which increases energy expenditure by elevating fat burning. However, treating the fat cells with drugs that interfere with the two enzymes restored sensitivity to catecholamine, allowing the fat cells to burn energy.Mowers et al. also treated obese mice with amlexanox, a drug that inhibits these enzymes, and found that this treatment made the mice sensitive to a synthetic catecholamine that triggered the release of energy from fat. Mowers et al. suggest, therefore, that IKKε and TBK1 respond to inflammation in the body by reducing catecholamine signaling, thus preventing energy expenditure. Drugs targeting these enzymes may be useful for treating conditions like obesity or type 2 diabetes.DOI:http://dx.doi.org/10.7554/eLife.01119.002 Obesity generates a state of chronic, low-grade inflammation in liver and adipose tissue accompanied by macrophage infiltration and the local secretion of inflammatory cytokines and chemokines that attenuate insulin action, resulting in insulin resistance and the subsequent development of Type 2 diabetes . NumerouThe transcription factor NFκB and its inflammatory program play an important role in the development of insulin resistance in obese liver and adipose tissue . NFκB isWe recently reported that both mRNA and protein expression levels of IKKε and TBK1 are increased in adipose tissue from mice fed a high fat diet . Both ofUcp1 gene expression after treatment with the non-selective β-adrenergic agonist, isoproterenol (ISO), or the β3-adrenergic agonist, CL-316,243. Fold difference in Ucp1 gene expression was calculated by normalization of relative Ucp1 mRNA levels in treated relative to control samples. Treatment of empty vector-expressing cells with ISO or CL-316,243 resulted in a 1.6-fold or twofold increase in Ucp1 mRNA levels, respectively  was upregulated, as previously reported  is the major PDE isoform expressed in adipocytes . Genetictabolism . Phosphotabolism .Since we observed that cAMP production was impaired in forskolin or isoproterenol-stimulated 3T3-L1 adipocytes overexpressing IKKε , we examin vitro with [γ-32P]ATP and purified PDE3B as a substrate. Phosphorylation was assessed by SDS-PAGE followed by autoradiography. TBK1 directly catalyzed the phosphorylation of PDE3B; phosphorylation was also produced by incubation with Akt and PKA, as previously reported (in vitro (data not shown). This increase in phosphorylation produced by in vitro incubation with TBK1, IKKε and PKA was also detected when PDE3B was blotted with antibodies that recognize the 14-3-3 binding motif  with anti-HA antibodies. Expression of IKKε in cells caused a shift in electrophoretic mobility of PDE3B, and this shift was not detected when IKKε K38A was expressed . PhosphoPrevious studies suggested that IKKε and TBK1 bind to their respective substrates through a sequence that includes a ubiquitin-like domain (ULD) proximal to their kinase domain. This domain is highly conserved among the IKK family members, and is 49% identical between IKKε and TBK1 . To confNext, to test further the role of PDE3B phosphorylation by IKKε and TBK1 in initiating its interaction with 14-3-3β, we prepared a GST-14-3-3β fusion protein which was incubated with lysates from HEK293T cells co-expressing TBK1 with PDE3B. PDE3B was preferentially pulled down by GST-14-3-3β after phosphorylation by TBK1 but not by its inactive K38A mutant, whereas GST beads alone enriched neither PDE3B nor its phosphorylated form .To evaluate the regulatory role of PDE3B phosphorylation by IKKε and TBK1, we determined which sites are phosphorylated. HA-PDE3B was co-expressed in Cos-1 cells with IKKε and TBK1, and phosphorylated PDE3B was enriched by IP with anti-HA antibodies. Phosphorylation sites on human PDE3B were then determined by LC-MS/MS mass spectrometry. This analysis revealed that serines 22, 299, 318, 381, 463, 467, and 503 were phosphorylated by both kinases; there were no differences between the kinases . InteresWhile several serine residues are known to be phosphorylated on PDE3B in response to stimuli, serine 318 (human) is the best characterized. This residue resides in a consensus phosphorylation sequence for both Akt and PKA, and also serves as a consensus 14-3-3 binding motif once phosphorylated . We thusTo examine the functional importance of the phosphorylation of PDE3B at Serine 318, we overexpressed WT PDE3B and its S318A mutant in 3T3-L1 adipocytes, and tested the response of the cells to TNFα. Overexpression of WT PDE3B in cells reduced the attenuation of forskolin-stimulated cAMP production and phosphorylation of HSL produced by TNFα, whereas PDE3B S318A was ineffective . These din vivo, we investigated whether the administration of a selective inhibitor of IKKε and TBK1, amlexanox, can reverse diet-induced catecholamine resistance in rodents. We fed mice a high fat or normal diet, treated them with amlexanox by oral gavage for 4 days (prior to the point when weight loss is seen), and then gave a single intraperitoneal (IP) injection of the β3-adrenergic agonist CL-316,243. Injection of CL-316,243 stimulated a threefold increase in serum FFA and glycerol levels in both vehicle and amlexanox-treated mice on normal diet (ND). The effect of CL-316,243 to increase serum FFAs was significantly attenuated in HFD-fed, vehicle-treated mice. However, HFD-fed mice treated with amlexanox responded like normal diet mice, despite the fact that they were weight matched with control HFD-fed mice  unless stated otherwise. Anti-Flag antibody was obtained from Sigma, and anti-HA antibody was obtained from Santa Cruz Biotechnology . Anti-IKKε, anti-TBK1, anti-phospho-TBK1 (Ser172), anti-AKT, anti-phospho-AKT (Ser473), anti-HSL, anti-phospho-HSL (Ser660), anti-p38, anti-phospho-p38, anti-perilipin, anti-ATGL and anti-PPARγ antibodies were purchased from Cell Signaling Technology . Anti-phospho-perilipin (Ser522) was purchased from Vala Sciences Inc . Anti-CGI-58 was purchased from Novus Biologicals . Anti-RalA antibody was obtained from BD Bioscience . Anti-Ucp1 antibody was obtained from Alpha Diagnostics . Anti-PDE3B was provided as a generous gift by the Dr Vince Manganiello . Enhanced chemiluminescence (ECL) reagents were purchased from Thermo Scientific . EDTA-free protease inhibitor tablet was purchased from Roche Diagnostics . Monoclonal anti-HA agarose (Sigma) was used for immunoprecipitations, performed using the manufacturer’s protocol. The human PDE3B cDNA was kindly provided by Dr Morris Birnbaum (University of Pennsylvania). The human 14-3-3β cDNA was kindly provided by Dr Ken Inoki (University of Michigan) and subcloned into pKH3  and pGEX3T3-L1 fibroblasts  were cultured and differentiated as described previously . The cel4P2O7, and freshly added protease inhibitor tablet), then incubated for 1 hr at 4°C . Samples were diluted in sodium dodecyl sulfate (SDS) sample buffer and boiled for 5 min at 95°C. Proteins were resolved by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes . Individual proteins were detected with the specific antibodies and visualized on film using horseradish peroxidase-conjugated secondary antibodies  and Western Lightning Enhanced Chemiluminescence .Tissues were homogenized in lysis buffer  starting at 8 weeks of age for up to 6 months, while normal diet C57BL/6 controls were maintained on normal chow diet consisting of 4.5% fat . Animals were housed in a specific pathogen-free facility with a 12-hr light/12-hr dark cycle and given free access to food and water. All animal use was in compliance with the Institute of Laboratory Animal Research Guide for the Care and Use of Laboratory Animals and approved by the University Committee on Use and Care of Animals at the University of Michigan.Adrp was chosen as the internal control for normalization as its expression was not significantly affected by experimental conditions. Sequences of Ucp1 primers used in this study are 5′-AGGCTTCCAGTACCATTAGGT‐3′ and 5′‐CTGAGTGAGGCAAAGCTGATTT‐3′. Sequences of Ikbke primers used in this study are 5′-ACAAGGCCCGAAACAAGAAAT-3′ and 5′-ACTGCGAATAGCTTCACGATG‐3′. Data were analyzed using the 2−ΔΔCT method  with a DNase step. The Superscript First-Strand Synthesis System for RT-PCR  was used with random primers for reverse transcription. Real-time PCR amplification of the cDNA was performed on samples in triplicate with Power SYBR Green PCR Master Mix  using the Applied Biosystems 7900HT Fast Real-time PCR System. T method .Mice were placed on a high fat diet for 6 months, then after 1 week of daily gavage with vehicle, mice were gavaged with either vehicle or amlexanox (25 mg/kg) daily for 4 days. On the fourth day, mice were injected with CL-316,243 (1 mg/kg) or saline control. For analysis of blood metabolites, serum samples were collected before and 15 min after the injection, via a submandibular bleed. Mice were euthanized and WAT samples were collected for cAMP measurement and western blot analysis, 15 min or 20 min after injection. Analysis of oxygen consumption was performed in metabolic cages, as previously described , by the For experiments involving CL-316,243, pieces were pre-incubated for 30 min with amlexanox (100 μM) or DMSO vehicle control; then the tissue pieces were transferred to fresh media with and without 10 mM CL-316,243, and media samples were collected every 15 min for 1 hr. To measure glycerol release, 10 μl of supernatant was combined with 200 μl of Free Glycerol Reagent from the Free Glycerol Determination Kit (Sigma) and allowed to incubate for 15 min at room temperature. Absorbance at 540 nm was measured to determine glycerol content and was normalized to determine glycerol production per mg of white adipose tissue.3T3-L1 adipocytes were incubated in DMEM (Invitrogen) without phenol red for 2 hr at 37°C. The cells then were incubated for 90 min at 37°C in HBSS-2% fatty acid-free BSA with 10 μM isoproterenol or 10 μM CL-316,243. Free glycerol concentration was measured by reacting 25 μl of conditioned media with 200 μl Free Glycerol Reagent (Sigma) and absorbance was measured at 540 nm using the manufacturer’s protocol. Glycerol release was normalized to cellular protein content.3T3-L1 adipocytes were treated with β-agonists and/or phosphodiesterase inhibitors and allowed to incubate for the indicated amount of time at 37°C. The cells were lysed with 150 μl 0.1 M HCl, scraped and spun down. A cAMP Enzyme Immunoassay Kit (Sigma CA201) was used to quantify cAMP levels. 50 μl of cell lysates was combined with 50 μl Assay Buffer 2 in each well and cAMP levels were assayed according to the manufacturer’s protocol. Tissue samples were homogenized in 5% TCA, then extracted with water-saturated ether, and dried before resuspension in Assay Buffer 2.80 μl of packed 3T3-L1 cells was electroporated with 3 μg of Glosensor 22-F using Amaxa Cell Line Nucleofector Kit L (Lonza) according to the manufacturer’s protocol. Electroporated cells were resuspended in 10 ml of L1-FBS media and 200 μl per well was plated in six columns of an opaque, white, 96-well tissue culture plate . After 20 hr, media were changed to 100 μl DMEM with 1.5 mg/ml luciferin and allowed to equilibrate for 1 hr. The cells were treated with 50 μM forskolin and luminescence was read every 30 s for 75 min.In vitro kinase assays were performed by incubating purified kinase  in kinase buffer containing 25 mM Tris (pH 7.5), 10 mM MgCl2, 1 mM DTT, and 10 μM ATP for 30 min at 30°C in the presence of 0.5 μCi γ-[32P]-ATP and 1 μg myelin basic protein (MBP) per sample as a substrate. IKKε and TBK1 were fused to MBP  and these fusion proteins were purified from insect SF9 cells by baculovirus expression system by Dr Stuart J Decker . IKKβ, PKA, and AKT kinases were purchased from Millipore . The kinase reaction was stopped by adding 4X sodium dodecyl sulfate (SDS) sample buffer and boiling for 5 min at 95°C. Supernatants were resolved by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and analyzed by autoradiography using a Typhoon 9410 phosphorimager . The bands were quantified using ImageQuant.Calf intestinal phosphatase (CIP) was obtained from New England Biolabs . Immunoprecipitated PDE3B was incubated for 1 hr at 37°C in a 100 μl reaction containing 50 mM Tris pH 7.5, 150 mM NaCl, 1% NP-40, EDTA-free protease, and 5 μl CIP.For assays requiring soluble protein, purified GST-14-3-3β protein was eluted from glutathione beads by washing beads with 10 mM glutathione in PBS, pH 8.0. The elution was monitored by A280 readings, and fractions containing protein were pooled and dialyzed overnight against 4 L of ice-cold PBS. The proteins were then concentrated using an Amicon centrifugal filtration unit (Millipore). Concentrated proteins were stored at −80°C in PBS containing 10% glycerol and 10 mM DTT.g for 10 min and then were incubated with ∼10 mg of GST or GST-14-3-3β bound to glutathione beads  for 1.5 hr at 4°C. For samples treated with phosphatase, lysates were preincubated with 500 U of calf intestinal phosphatase  at 37°C for 1 hr before adding GST-14-3-3 beads. The beads were washed three times with 1 ml of PD buffer and then resuspended in 2X SDS sample buffer.For GST-14-3-3 pulldowns, cells were washed twice with ice-cold PBS and then lysed in 1 ml of 14-3-3 pulldown buffer  supplemented with a protease inhibitor tablet (Roche). Lysates were cleared by centrifuging at 13,000×GST or GST-14-3-3β was labeled with DIG by incubating 25 mg of protein with 2 ml of 5 mM Digoxigenin-3-O-methylcarbonyl-ε-aminocaproic acid-N-hydroxysuccinimide ester  in 350 ml of PBS for 15 min at room temperature. The labeling reaction was stopped by adding 100 ml of 1 M Tris-HCl, pH 7.4. Labeled protein was dialyzed against 1 L of 25 mM Tris-HCl, pH 7.4 for 1 hr at room temperature, then against 4 L of PBS, pH 7.4 for 4 hrs at 4°C, and finally against 4 L of fresh PBS, pH 7.4 for 16 hrs at 4°C. Labeled protein was then diluted in 25 ml of TBS  containing 2 mg/ml BSA (Sigma-Aldrich) and 0.01% sodium azide (Sigma-Aldrich). DIG-labeled proteins were stored at 4°C.For overlay assays, PDE3B was immunoprecipitated from HEK293T cells and resolved by SDS-PAGE. Proteins were transferred to a nitrocellulose membrane. The membrane was blocked at room temperature overnight in blocking buffer (5% skim milk in TBS-T). The membrane was then incubated with DIG-labeled proteins for at least 4 hr at 4°C and then washed three times with TBS-T. The membrane was then incubated with blocking buffer containing anti-DIG HRP antibody  for 2 hr at room temperature, and washed three times with TBS-T. Overlays were visualized by reacting with ECL western blotting substrate .In-gel digestion followed by LC-MS/MS analysis was carried out by the mass spectrometry-based proteomics resource in the Department of Pathology, University of Michigan. Briefly, tryptic peptides were resolved on a nano-C18 reverse phase column and sprayed directly onto Orbitrap mass spectrometer . Orbitrap was operated in a data-dependent mode to acquire one full MS spectrum  followed by MS/MS spectra on six most intense ions (top 6). Proteins were identified by searching data against human protein database  using X!Tandem/TPP software suite. Oxidation of Met, carbamidomethylation of Cys and phosphorylation of Ser, Thr, and Tyr were considered as potential modifications .t test to determine statistical significance. When more than two groups and two factors were investigated, we first performed a two-way analysis of variance (ANOVA) to establish that not all groups were equal. After a statistically significant ANOVA result, we performed between-group comparisons using the Tukey post hoc analysis for comparisons of all means and Sidak for comparisons of within factor main effect means. ANOVA and Tukey/Sidak tests were performed using GraphPad Prism version 6.Averaged values are presented as the mean ± SEM. When comparing two groups, we performed Student’s  review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  Lipolysis was determined as glycerol release per g of adipose tissue. This is problematic because adipose tissue mass and adipocyte size changes in response to high fat feeding. The “decrease” in lipolytic rates  may result from increased TAG accumulation and adipocyte mass rather than from reduced lipase activities. Glycerol release should be displayed per cell or per DNA content and not tissue mass.2) Also, glycerol release is a compound value of TAG hydrolysis and re-esterification of glycerol intermediates. In order to determine whether lipase activities are affected, it is necessary to measure their actual activities in in vitro assays.3) The increase in lipolysis in response to ISO and FSK are surprisingly moderate  and glycerol release data correlate relatively poorly to P-HSL levels .4) To strengthen the data on lipolysis the authors should analyze perilipin phosphorylation and the expression of ATGL/CGI-58. Particularly perilipin phosphorylation by PKA is a strong determinant of lipolysis.5) Figure 2A: Why does UCP1 expression not return to the level of empty vector when IKKe is mutated (IKKe K38A)? What happens to UCP1 expression in TNFa treated 3T3-L1 cells?6) Throughout all figures the induction of HSL phosphorylation in response to ISO and FSK is very inconsistent. Additionally, glycerol release and cellular cAMP concentrations do not correlate well with HSL phosphorylation  in the TNF experiments – why? What are the long-term TNF-treated phosphorylation patterns of HSL and perilipin?7) Figure 4C lacks a true negative control of a kinase that does not phosphorylate PDE3B.8) In several instances, the sample number is insufficient to provide statistically meaningful information. For example, the authors mentioned that in Figure 1A isoproterenol-induced glycerol release in HFD is not significant due to small sample size (n=2). The sample numbers need to be increased in order to make defined conclusions.9) Isoproterenol induced glycerol release in ND IKKε KO is significantly enhanced compared to ND IKKε WT . However there were no differences in phospho-PKA substrate phosphorylation  and relative pHSL/HSL ratio . In contrast isoproterenol-induced phospho-PKA substrate phosphorylation  and relative pHSL/HSL ratio  were significantly increased in HFD IKKε KO compared to HFD IKKε WT, although there was no difference in isoproterenol-induced glycerol release in HFD IKKε KO and HFD IKKε WT . How can the authors interpret these apparent inconsistencies? Is this due to the lack of statistical significance due to the low sample size?10) To study β-adrenergic signaling several agonist were selectively employed isoproterenol, forskolin, CL-316,243. It would have been preferable if more consistency in the agonist used were reported. For example, either isoproterenol-induced UCP1 expression or CL-316,243-induced glycerol release should be provided in either Figure 2A or Figure 2B, respectively, for completeness.11) The data presented in Figure 3 clearly demonstrates that TNF-α induced IKKε in adipocytes suppresses cAMP and glycerol production, and that the IKKε and TBK1 inhibitors amlexanox and CAY10576 rescue the TNF-α induced defects. Since these are also pharmacological manipulations, it would be nice to have a genetic conformation, such as examining the effect of IKKε and TBK1 knockdown on TNF-α-mediated cAMP and glycerol production. 1) Lipolysis was determined as glycerol release per g of adipose tissue. This is problematic because adipose tissue mass and adipocyte size changes in response to high fat feeding. The “decrease” in lipolytic rates  may result from increased TAG accumulation and adipocyte mass rather than from reduced lipase activities. Glycerol release should be displayed per cell or per DNA content and not tissue mass.The reviewers bring up a good point, which reveals a fundamental problem in comparing these mouse models in which the weights are different. Although ?-adrenergic signaling and lipolysis are enhanced in white adipose tissue from IKK? knockout mice fed a high-fat diet, the body weights of IKK? knockout mice are reduced compared to WT mice. We realized that conclusions derived from these data are thus confusing and somewhat problematic. Because weight loss alone has the potential to improve catecholamine sensitivity in obese mice, it is impossible to determine from these data whether the differences were due to weight or direct IKK? effects. Furthermore, we now know that in addition to IKKε TBK1 also plays an important role in the regulation of the lipolytic pathway.Because of these problems in interpretation of the data, we have elected to remove Figure 1 in its entirety from the revised paper. However, in order to address the issue of the direct role of IKK? and TBK1 in energy expenditure and catecholamine sensitivity in vivo, we have added another experiment (2) Also, glycerol release is a compound value of TAG hydrolysis and re-esterification of glycerol intermediates. In order to determine whether lipase activities are affected, it is necessary to measure their actual activities in in vitro assays.FEBS J, 2009) and many others). The reason for this discrepancy is that in addition to its modest effect on catalytic activity, HSL (and perilipin) phosphorylation induces a translocation of the enzyme to the lipid droplet, where it can gain access to its substrate. This translocation effect relies on phosphorylation, and is probably the more important event in controlling activity in cells . Because of this, HSL activity per se is rarely measured in vitro after cell lysis.While it is relatively easy to demonstrate changes in lipolysis after treatment of adipocytes with beta-adrenergic agonists, the corresponding increase in lipase activity as measured in vitro is rarely more than 1.5 fold. Even when HSL is phosphorylated in vitro with recombinant PKA, the change in catalytic activity is relatively modest  phosphorylation are sufficient to make the point.3) The increase in lipolysis in response to ISO and FSK are surprisingly moderate  and glycerol release data correlate relatively poorly to P-HSL levels .Because of the reasons explained above, we have removed Figure 1.4) To strengthen the data on lipolysis the authors should analyze perilipin phosphorylation and the expression of ATGL/CGI-58. Particularly perilipin phosphorylation by PKA is a strong determinant of lipolysis.Endocrinology, 2006, Arimura et al., J Biol Chem., 2004) because TNF? treatment resulted in a dose-dependent decrease in PPARγ expression in 3T3-L1 adipocytes, as previously reported . However, expression of ATGL and CGI-58 were not changed in response to long-term TNFα treatment.As requested, we examined levels of phospho-perilipin using an anti-phospho-perilipin antibody that recognizes Ser522, which is a site phosphorylated by PKA . Perilip5) Figure 2A: Why does UCP1 expression not return to the level of empty vector when IKKe is mutated (IKKe K38A)? What happens to UCP1 expression in TNFa treated 3T3-L1 cells?Ucp1 gene expression in response to β-adrenergic stimuli in 3T3-L1 adipocytes overexpressing empty vector or IKKε, or IKKε K38A by electroporation. Interestingly, as shown before in numerous experiments, overexpression of IKKε K38A still produced a small reduction in the sensitivity of cells to β-adrenergic stimuli regarding Ucp1 expression  could result in activation of the complex. Thus, dimerization of IKKε K38A with an endogenous active kinase may produce increased activity of the complex. We have discussed this possibility in the revised paper.This is an interesting question and points to the limits in interpreting experiments in which the kinase inactive mutants are overexpressed. Firstly, the levels of protein achieved by overexpression of IKK? K38A are consistently less than those seen with the WT protein , probablpression , glyceropression  or cAMP pression  comparedpression , and botUcp1 expression in response to isoproterenol or forskolin, or CL-316,243  Throughout all figures the induction of HSL phosphorylation in response to ISO and FSK is very inconsistent. Additionally, glycerol release and cellular cAMP concentrations do not correlate well with HSL phosphorylation  in the TNF experiments – why? What are the long-term TNF-treated phosphorylation patterns of HSL and perilipin?We repeated our experiments to examine the effects of long-term TNFα treatment on phosphorylation of HSL and perilipin, and now include this new data in the paper. We observed that phosphorylation of HSL and perilipin correlated well with glycerol release and cellular cAMP levels .7) Figure 4C lacks a true negative control of a kinase that does not phosphorylate PDE3B.As a negative control, we performed in vitro kinase assay using recombinant IKKβ with purified PDE3B as a substrate, since this IKK is most closely related to TBK1 and IKKε. PDE3B phosphorylation was barely detected with IKKβ .8) In several instances, the sample number is insufficient to provide statistically meaningful information. For example, the authors mentioned that in Figure 1A isoproterenol-induced glycerol release in HFD is not significant due to small sample size (n=2). The sample numbers need to be increased in order to make defined conclusions.Because of the reasons explained above, we have removed Figure 1.9) Isoproterenol induced glycerol release in ND IKKε KO is significantly enhanced compared to ND IKKε WT . However there were no differences in phospho-PKA substrate phosphorylation  and relative pHSL/HSL ratio . In contrast isoproterenol-induced phospho-PKA substrate phosphorylation  and relative pHSL/HSL ratio  were significantly increased in HFD IKKε KO compared to HFD IKKε WT, although there was no difference in isoproterenol-induced glycerol release in HFD IKKε KO and HFD IKKε WT . How can the authors interpret these apparent inconsistencies? Is this due to the lack of statistical significance due to the low sample size?Because of the reasons explained above, we have removed the previous Figure 1.10) To study β-adrenergic signaling several agonist were selectively employed isoproterenol, forskolin, CL-316,243. It would have been preferable if more consistency in the agonist used were reported. For example, either isoproterenol-induced UCP1 expression or CL-316,243-induced glycerol release should be provided in either .3-adrenergic agonist, CL-316,243) and one beyond the receptor  to emphasize the role of noncanonical kinases IKKε and TBK1 on catecholamine resistance beyond the receptor level. Since differentiated 3T3-L1 adipocytes predominantly expresses the β3-adrenergic receptor , CL-316,243 was also used in some experiments. As requested, we now show isoproterenol or CL-316,243-induced UCP1 expression  The data presented in .Nat Med., 2013), even under knockdown conditions. The data below shows that we get ∼ 80% knockdown of TBK1 and ∼ 50% knockdown of IKKε in 3T3-L1 adipocytes by electroporation, but that 24 hours of TNFα treatment induces IKKε protein levels almost back to control levels, and also decreases PPARγ levels  in a dose dependent manner.This was an excellent idea and we pursued it. Unfortunately, it was not possible to achieve complete knockdown of IKKε and TBK1. Making matters more difficult, TNFα also induces both mRNA and protein levels of IKKε through NFκB activation (Reilly and Chiang et al.,


However, reversal of these two processes alone does not account for the force inhibition that occurs during relaxation or inhibition of contraction, implicating that other mechanisms, such as actin cytoskeletal rearrangement, play a role in the suppression of force. In this study, we hypothesize that forskolin-induced force suppression is dependent upon changes in actin cytoskeletal dynamics. To focus on the actin cytoskeletal changes, a physiological model was developed in which forskolin treatment of intact porcine coronary arteries (PCA) prior to treatment with a contractile agonist resulted in complete suppression of force. Pretreatment of PCA with forskolin suppressed histamine-induced force generation but did not abolish [Ca2+]i rise or MLC phosphorylation. Additionally, forskolin pretreatment reduced filamentous actin in histamine-treated tissues, and prevented histamine-induced changes in the phosphorylation of the actin-regulatory proteins HSP20, VASP, cofilin, and paxillin. Taken together, these results suggest that forskolin-induced complete force suppression is dependent upon the actin cytoskeletal regulation initiated by the phosphorylation changes of the actin regulatory proteins and not on the MLC dephosphorylation. This model of complete force suppression can be employed to further elucidate the mechanisms responsible for smooth muscle tone, and may offer cues to pathological situations, such as hypertension and vasospasm.Initiation of force generation during vascular smooth muscle contraction involves a rise in intracellular calcium ([Ca This increase in intracellular calcium activates calmodulin-dependent myosin light chain kinase (MLCK), leading to increases in the phosphorylation of 20 KDa myosin light chains (MLC). Crossbridge phosphorylation of the actomyosin apparatus results in the generation of force in vascular smooth muscle 2+ and MLC phosphorylation are important for the initiation of contraction, the tonic phase, or force maintenance, of smooth muscle contraction can occur where [Ca2+]i levels and MLC phosphorylation are near basal levels, suggesting other pathways are engaged during force maintenance in smooth muscle 2+, crossbridge phosphorylation, and shortening velocity fall to intermediate values Smooth muscle activation of the H1 receptor via histamine is linked to the intracellular G protein (Gαq) and Gαq-coupled receptors that activate phospholipase C (PLC) and RhoA  or adenylyl cyclase , leading to increases in cGMP and cAMP, respectively. The cyclic nucleotides, in turn, activate cGMP-dependent protein kinase (PKG) and cAMP-dependent protein kinase (PKA) 2+]i were measured concurrently using a FluoroPlex Tissue Bath Fluorometry System, and polymerization of actin, phosphorylation of actomyosin and actin-associated proteins were determined. Our results indicate that forskolin-induced suppression of force does not completely abolish Ca2+ transients or MLC phosphorylation but causes depolymerization of actin dose-dependently, and changes in the phosphorylation of proteins such as HSP20, cofilin, VASP and paxillin that regulate actin polymerization. Since force can be completely suppressed without abolishing [Ca2+]i, this model system can be employed to elucidate as yet unidentified Ca2+-independent molecular determinants of force inhibition in vascular smooth muscle.In this study, we hypothesized that cyclic nucleotide-induced force suppression is dependent upon changes in actin cytoskeletal dynamics. We developed a physiological model in which histamine-induced force was completely suppressed by forskolin pretreatment in PCA smooth muscle. In this novel approach, force and -1-propanesulfonate) were from Research Organics Inc. . F/G Actin assay kit was from Cytoskeleton Inc., . Fura 2-AM and Pluronic F-127 was purchased from Invitrogen .4, 1.0 mM NaH2PO4, 1.5 mM CaCl2, 10 mM glucose, and 10 mM HEPES, pH 7.4), and the coronary arteries were dissected and tested immediately or after overnight storage at 4°C in University of Wisconsin solution. Subcutaneous fat and adventitial tissues were removed and the vessel was cut into transverse rings of 3.0 mm in width. To focus on the smooth muscle-derived changes during inhibition of force, endothelium was denuded by gently rolling the luminal surface of each ring at the tip of a fine forceps. Rings were suspended in a muscle bath containing a bicarbonate buffer , equilibrated with 95% O2/5% CO2, at 37°C. Force measurements were obtained with either a Kent Scientific  force transducer (TRN001) or a Radnoti force transducer  interfaced with Power Lab from AD Instruments . Data were recorded with Chart software, version 5.1.1 (AD Instruments). Rings were washed every 15 min with 37°C bicarbonate buffer for 1 hr, and each ring was progressively stretched to its optimal resting tension (approximately 1 g) that would produce a maximal response to contractile agonists as determined previously, then maintained at the resting tension and equilibrated for another hour. Rings were then contracted multiple times with high extracellular potassium  and the force generated was measured. Measured force was normalized for ring weight and length and converted to Stress using the formula: Stress [105Newtons (N)/m2] = force (g) × 0.0987/area, where area is equal to the wet weight [mg/length ] divided by 1.055. The maximal tension obtained was taken as 100%. Rings were equilibrated for an additional 30 min and dose response curves for histamine contraction and forskolin relaxation were determined to select the correct dose of agents for the experiment. To determine the inhibition of contraction, rings were either treated with buffer alone (control), histamine (5 µM ) for 3 min, forskolin (5 µM) for 10 min, or forskolin  for 10 min followed by histamine (5 µM) for 3 min. At the end of the experiments, all rings were washed and contracted with KCl to ensure continued viability of the tissues. To determine the role of phosphorylation of proteins during inhibition of force, physiologic experiments were conducted as described above and the tissues were snap frozen under tension using forceps precooled in liquid nitrogen at 3 min and then pulverized. These pulverized tissues were stored at −80°C for later analysis using urea glycerol gel, SDS polyacrylamide gel electrophoresis (PAGE) or isoelectric focusing and western blotting. For actin assay to determine the level of F-actin compared to G-actin, the tissues were used immediately after treatment without freezing.Fresh cadaveric hearts were isolated immediately from euthanized, discarded animals from Vanderbilt University Medical Center. All procedures for collection of cadaveric tissue were reviewed and approved by the Vanderbilt University Animal Care and Use Committee. Porcine hearts were also collected from the local slaughter house  after obtaining permission from the slaughter house to use the tissue for research. The heart was procured and placed directly in HEPES buffer , followed by histamine (5 µM) or histamine alone. To add forskolin or histamine while continuously measuring intracellular calcium concentrations, an infusion line filled with bicarbonate buffer was used to keep the system in a closed light impenetrable state. The amount of buffer in the infusion line was adjusted to achieve the final concentration of the agonist in the bath. Force and calcium fluorescence were measured continuously for 15 min after the addition of histamine.Rings of PCA were suspended on hooks in a FluoroPlex Tissue Bath Fluorometry System , which enables fluorescence ion recording in parallel with force measurement. Force measurements were obtained with a Radnoti force transducer  interfaced with Power Lab from AD Instruments . Rings were loaded at room temperature with 10 µM Fura-2 AM ester and 0.01% Pluronic F-127 in the bicarbonate buffer for 4 hrs. After loading, rings were washed every 10 min with 37°C bicarbonate buffer for 1 hr. i, MLC phosphorylation, F-actin levels and HSP20 phosphorylation were measured. Pretreatment of PCA with forskolin at 1 µM did not abolish histamine-induced force    for tissue that was treated with histamine alone compared to forskolin followed by histamine treatment . Forskolin at 5 µM did not significantly change MLC phosphorylation    A, E. Ch, n = 7)  B, E, ML, n = 7)  or signi, n = 7) . However, n = 7) . Forskol, n = 7)  but did , n = 9)  C, E. Th, n = 9) . The tim, n = 7) . Forskol, n = 7) , and F-a, n = 7)  while it, n = 7) . Hence, , n = 7) , and demHistamine treatment significantly increased the phosphorylation of MLC  when compared to untreated basal  . Forskolin plus histamine treatment also significantly increased . There was no significant difference in the MLC phosphorylation between histamine and forskolin plus histamine treated tissues , which sSeveral investigators have demonstrated that actin is polymerized during contraction of smooth muscle, and agents that inhibit actin polymerization result in inhibition of contraction , suggesting that the decoupling between [Ca2+]i and force was not complete. It is possible that these changes may be due to the direct effect of forskolin on the [Ca2+]i. The effect was dose-dependent as the lower forskolin concentrations (1 µM) did not abolish the transient [Ca2+]i or the force, and at a higher concentration (10 µM) there was no transient increase in [Ca2+]i and force was completely suppressed. These results suggest that a higher forskolin dose inhibits the agonist-induced Ca2+ signaling pathways, possibly due to cross activation of PKG by cAMP, as reported by several investigators 2+]i and the force developed for a given intracellular [Ca2+]  2+]i and force but also decreased the phosphorylation of MLC .While histamine-induced force generation was completely suppressed with forskolin pretreatment, a transient increase in i or MLC phosphorylation. Actin depolymerization creates conditions in which there is no actin filament structure on which MLC can treadmill. Thus, calcium desensitization is likely not the only mechanism of force modulation during activation of cyclic nucleotide-dependent signaling pathways. Our results further suggest that the actin cytoskeletal changes are mediated by the phosphorylation changes of actin modulatory proteins, such as HSP20, cofilin, VASP and paxillin. The force suppression model employed in this study can be used to further characterize the role of actin and its associated proteins in the regulation of vascular smooth muscle tone. Future studies may also examine later events (3–30 min) of force maintenance. In addition, cyclic nucleotide analogues can be used to decipher the different mechanisms that contribute to these regulatory pathways.A physiological model was developed in which forskolin pretreatment completely suppressed histamine-induced force in PCA by regulating actin polymerization and dynamics without abolishing increases in [Ca