MRP Proteins Research Articles

MrpA (all1838), a gene involved in alkali and Na + sensitivity, may also have a role in energy metabolism in the cyanobacterium Anabaena sp. strain PCC 7120

Anabaena sp. PCC7120 contains a gene, mrpA (all1838), which forms part of a seven gene-cluster (all1843-all1837) with significant sequence similarity to bacterial operons that putatively code for a multicomponent cation/proton antiporter involved in alkaline pH adaptation and salt resistance. We previously showed that growth and photosynthesis were inhibited in a strain mutated in mrpA, denoted as PHB11, particularly at alkaline pH. Here, we show that respiration was also impaired in the mutant independently of the external pH. In addition, at high pH, less ATP and vegetative cell ferredoxin were present in PHB11, which also showed lower levels of ferredoxin-NADP(+) oxidoreductase (FNR). Ferredoxin and FNR are involved in the generation of reductant NADPH in cyanobacteria. These results suggest an energetic role of mrpA (and perhaps of the whole mrp-gene cluster) in Anabaena sp. PCC 7120 that is further supported by the significant similarity of putative Anabaena Mrp proteins to membrane subunits of complex I.

The MrpA, MrpB and MrpD subunits of the Mrp antiporter complex in Bacillus subtilis contain membrane-embedded and essential acidic residues

Bacillus subtilis Mrp is a unique Na+/H+ antiporter with a multicomponent structure consisting of the mrpABCDEFG gene products. We have previously reported that the conserved and putative membrane-embedded Glu-113, Glu-657, Asp-743 and Glu-747 of MrpA (ShaA) are essential for the transport function. In this study, we further investigated the functional involvement of the equivalent conserved acidic residues of other Mrp proteins in heterologous Escherichia coli and natural B. subtilis backgrounds. Asp-121 of MrpB and Glu-137 of MrpD were additionally identified to be essential for the transport function in both systems. Glu-137 of MrpD and Glu-113 of MrpA were found to be conserved in the homologous MrpD/MrpA proteins as well as in the homologous subunits of H+-translocating primary active transporters such as Nuo and Mbh, suggesting their critical role in ion binding. The remaining essential acidic residues clustered in the C-terminal domain of MrpA (Glu-657, Asp-743 and Glu-747) and MrpB (Asp-121); these subunits are fused in some Gram-negative species. It is possible that the MrpA, MrpB and MrpD subunits, which contain essential transmembrane acidic residues, form the ion translocation site(s) of the Mrp antiporter complex.

Determination Of The Lipid Membrane Composition Of J774 Macrophages Cells Surexpressing Mrp Protein (resistant To Ciprofloxacin)

Ciprofloxacin (CIP) is a fluoroquinolone antibiotic with an activity towards both extracellular and intracellular bacteria (Seral et al., 2005). Diffusion and efflux processes modulate accumulation of this drug within eukaryotic cells. When J774 macrophages were grown in presence of ciprofloxacin, the antibiotic is subject to constitutive efflux through the activity of an MRP-related transporter (Michot et al., 2004).In view of the critical role of lipids for both drug uptake and activity of MRP proteins (Hinrichs et al, 2004), together with the ability of fluoroquinolones to interact with lipids (Bensikaddour et al., 2008 (a,b)), we investigated the composition of lipids in resistant and sensitive J 774 macrophages to ciprofloxacin.Firstly, we characterized by thin layer chromatography the phospholipids composition of J774 macrophages cells sensitive (WT) and resistant to ciprofloxacin (CIP). Results showed that sphingomyelin (SM) decreased 2 times whereas phosphatidylinositol increased 1.5 fold in resistant cells. Phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and cholesterol didn't show any significant change. Secondly, we studied membrane fluidity of liposomes mimicking WT and CIP resistant J774 cells, by fluorescence polarization spectroscopy. In this respect, we observed a decrease in the melting temperature in vesicles mimicking the membrane composition of CIP resistant cells, indicating that changes in the fluidity of these membranes may be due to the decrease of SM. Studies are currently performed to investigate potential changes in other components of rafts like glucosylceramides. These data might have important relevance to relate the interaction of fluoroquinolones with lipids and change in the processes involved in cellular accumulation of fluoroquinolones.

In vitro evaluation of the effects of gefitinib on the cytotoxic activity of selected anticancer agents in a panel of human endometrial cancer cell lines

This study was conducted to determine the in vitro optimal combination of selected anticancer agents with gefitinib and evaluate its effect on the expression of correlative biological targets in the cell-signaling pathway. In addition, the effect of gefitinib on the expression of ATP-binding cassette (ABC) transport proteins was evaluated. Growth inhibition assays were conducted in six human endometrial cancer cell lines to evaluate the activity of selected anticancer agents with gefitinib compared to each alone. Enzyme linked immunosorbant assay (ELISA) assessed the presence of pEGFR in treated and untreated cells. Evaluation of the suppression of correlative biological targets in the cell-signaling pathway was completed by immunoblotting. RT-PCR was used to characterize the expression of MRP and ABC transport proteins. This in vitro study gefitinib did not observe cytotoxic activity as a single agent. However, the activity of gefitinib as EGFR inhibitor was confirmed. The combination of gefitinib with paclitaxel and docetaxel exhibited improved in vitro cytotoxic activity compared to each antineoplastic agent alone. Suppression of pAKT and p27 in the human endometrial cancer cells treated with selected combinations of chemotherapeutic drugs and gefitinib was observed. These data suggest that EGFRinhibitors, such as gefitinib, have the potential to modulate common mechanisms of drug resistance and may have a role in optimizing antineoplastic regimens for the treatment of recurrent endometrial cancer. This may represent a promising option for this class of agents in the treatment of endometrial cancer.

Single Gene Deletions of mrpA to mrpG and mrpE Point Mutations Affect Activity of the Mrp Na + /H + Antiporter of Alkaliphilic Bacillus and Formation of Hetero-Oligomeric Mrp Complexes

Mrp antiporters catalyze secondary Na(+)(Li(+))/H(+) antiport and/or K(+)/H(+) antiport that is physiologically important in diverse bacteria. An additional capacity for anion flux has been observed for a few systems. Mrp is unique among antiporters in that it requires all six or seven hydrophobic gene products (MrpA to MrpG) of the mrp operon for full antiporter activity, but MrpE has been reported to be dispensable. Here, the membrane complexes formed by Mrp proteins were examined using a cloned mrp operon from alkaliphilic Bacillus pseudofirmus OF4. The operon was engineered so that the seven Mrp proteins could be detected in single samples. Membrane extracts of an antiporter-deficient Escherichia coli strain expressing this construct were analyzed by blue native-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mrp complexes of two sizes were identified containing all seven Mrp proteins. Studies of the single nonpolar mrp gene deletions in the construct showed that a subcomplex of MrpA, MrpB, MrpC, and MrpD was formed in the absence of MrpE, MrpF, or MrpG. By contrast, MrpE, MrpF, and MrpG were not observed in membranes lacking MrpA, MrpB, MrpC, or MrpD. Although MrpA and MrpD have been hypothesized to be the antiporter proteins, the MrpA-to-D complex was inactive. Every Mrp protein was required for an activity level near that of the wild-type Na(+)/H(+) antiporter, but a very low activity level was observed in the absence of MrpE. The introduction of an MrpE(P114G) mutation into the full Mrp complex led to antiport activity with a greatly increased apparent K(m) value for Na(+). The results suggested that interactions among the proteins of heterooligomeric Mrp complexes strongly impact antiporter properties.

Renal and Hepatic Transporter Expression in Type 2 Diabetic Rats

Membrane transporters are critical for the uptake as well as elimination of chemicals and by-products of metabolism from the liver and kidneys. Since these proteins are important determinants of chemical disposition, changes in their expression in different disease states can modulate drug pharmacokinetics. The present study investigated alterations in the renal and hepatic expression of organic anion and cation transporters (Oats/Octs), multidrug resistance-associated proteins (Mrps), breast cancer resistance protein (Bcrp), P-glycoprotein (Pgp), and hepatic Na(+)-taurocholate cotransporting polypeptide (Ntcp) in type 2 diabetic rats. For this purpose, type 2 diabetes was induced by feeding male Sprague-Dawley rats a high fat diet followed by a single dose of streptozotocin (45 mg/kg, i.p., in 0.01 M citrate buffer pH 4.3) on day 14. Controls received normal diet and vehicle. Kidney and liver samples were collected on day 24 for generation of crude plasma membrane fractions and Western blot analysis of Oat, Oct, Mrp, Bcrp, Pgp, and Ntcp proteins. With regards to renal uptake transporters, type 2 diabetes increased levels of Oat2 (2.3-fold) and decreased levels of Oct2 to 50% of control kidneys. Conversely, efflux transporters Mrp2, Mrp4, and Bcrp were increased 5.4-fold, 2-fold, and 1.6-fold, respectively in type 2 diabetic kidneys with no change in levels of Mrp1, Mrp5, or Pgp. Studies of hepatic transporters in type 2 diabetic rats reveal that the protein level of Mrp5 was reduced to 4% of control livers with no change in levels of Bcrp, Mrp1, Mrp2, Mrp4, Ntcp, or Pgp. The changes reported in this study may have implications in type 2 diabetic patients.

Reversal of P-Glycoprotein-Mediated Drug Efflux by Eudesmin from Haplophyllum perforatum and Cytotoxicity Pattern versus Diphyllin, Podophyllotoxin and Etoposide

The present study focuses on eudesmin (bicyclic lignan, 0.15 % of dry leaves) and diphyllin (arylnaphthalene lignan, 0.1 % of dry roots), both isolated from H. perforatum Kar. et Kir, a Rutaceae species endemic to Uzbekistan. We first compared their specificity for cancer cells with those of etoposide and podophyllotoxin by screening their cytotoxicity on 3 healthy cell-lines and 7 sensitive or resistant human solid cancer lines. We then tested their capacity to reverse P-glycoprotein-mediated multidrug resistance (MDR) by assaying dye and drug uptake in MDR1-transfected Madin-Darby canine kidney (MDCK-MDR1) and doxorubicine-resistant human breast carcinoma cells (MCF7/Dox). Eudesmin displays IC (50) values > 100 microM on all tested lines. Our data provide the first demonstration that this non-toxic lignan reverses Pgp-mediated drug efflux and supports the hypothesis that it may inhibit resistance mediated by MDR1 and MRP proteins. Even if its reversal activity is insufficient for clinical application, its capacity to accumulate [(3)H]-vinblastine in MDCK/MDR1 and MCF7/Dox cells suggests that eudesmin may positively affect the bioavailability and, thereby, the therapeutic potency of anticancer drugs in Pgp-overexpressing cells. Diphyllin exhibits IC (50) values ranging from 10 (- 6) to 10 (- 4) M. It is markedly less toxic than podophyllotoxin (IC (50) : 13 - 61 nM), but exhibits tumoricidal effects close to those of etoposide. Unfortunatly, it is 65-fold more toxic than etoposide on human primary fibroblasts. Consequently, it has no value as an anticancer drug. Its value as raw material for the hemisynthesis of anticancer drugs is discussed.

Complex Formation by the mrpABCDEFG Gene Products, Which Constitute a Principal Na + /H + Antiporter in Bacillus subtilis

The Bacillus subtilis Mrp (also referred to as Sha) is a particularly unusual Na(+)/H(+) antiporter encoded by mrpABCDEFG. Using His tagging of Mrp proteins, we showed complex formation by the mrpABCDEFG gene products by pull-down and blue native polyacrylamide gel electrophoresis analyses. This is the first molecular evidence that the Mrp is a multicomponent antiporter in the cation-proton antiporter 3 family.

A novel role of myeloid related protein in dendritic cell differentiation (49.11)

Abstract Abnormal differentiation of dendritic cell (DC) is a critical factor contributing into ineffective immune response in cancer. We have searched for the genes that could be involved into tumor-induced defects in DC differentiation and found that tumor-derived factors consistently up-regulated expression of myeloid related protein 14 (MRP14) and its partner MRP8 in hematopoietic progenitor and myeloid cells during DC differentiation. Normal DC differentiation was associated with dramatic down-regulation of MRP14/8, whereas accumulation of immature myeloid cells was associated with persistently high levels of these proteins. Overexpression of these proteins in embryonic stem cells resulted in inhibition of DC differentiation and accumulation of immature myeloid cells able to form myeloid colonies. It is known that Gr-1+ myeloid-derived suppressor cells (MDSC) frequently accumulate in tumor-bearing mice and during in vitro DC differentiation in the presence of tumor-derived factors. We have found that in tumor-bearing mice MRP14/8 were exclusively accumulated in MDSC. Accumulation of these proteins was controlled by up-regulation of STAT3. MRP14/8 knockout mice had normal level of DCs or MDSC suggesting that MRP proteins were not required for accumulation of MDSC in cancer. Thus, this study demonstrates that tumor-induced up-regulation of MRPs inhibits DC differentiation, which may suggest a novel molecular mechanism of tumor-induced abnormalities in myeloid cells in cancer.

Twenty years of ATP-binding cassette (ABC) transporters

ATP-binding cassette (ABC) transporters constitute a large superfamily of primary active transport systems, which play a diversity of physiological roles, and are found in all cells of all kingdoms [3]. The year 2006 marks the 20th anniversary of the identification of this superfamily of proteins in 1986 [4]. It is also the 30th anniversary of the molecular identification of P-glycoprotein (MDR1, ABCB1) [5], which, when its gene was sequenced in 1986, turned out to be the first mammalian example of the ABC transporter family. It seems appropriate to celebrate these milestones in ABC transporter research with a special issue. It is the aim of this issue to give an overview on current knowledge and novel findings on mammalian ABC proteins, highlighting the many achievements of 30 years of research in the field. It complements a special issue on ABC transporters in FEBS Letters, which appeared earlier this year [2]. In mammals, seven families of ABC transporter genes have been defined, coding for 48 individual transporters. Although the number of mammalian ABC proteins is much smaller than found in prokaryotes many are of major clinical significance; currently, 18 human ABC genes have been associated with genetic diseases [1]. All ABC transporters consist of four core domains [4]: two transmembane domains, each normally consisting of, most frequently, six transmembrane alpha-helices (TMD1 and TMD2), and two nucleotide binding domains (NBD1 and NBD2). These four core domains form the minimal functional unit. However, there are many variations on this theme. For example, the four domains of mammalian ABC transporters may be fused together in a number of ways, either as a single polypeptide or as two half transporters each consisting of one TMD and one NBD. The MRP (ABCC1, ABCC2, ABCC3, ABCC6, ABCC8, ABCC9, and ABCC10) proteins have an additional transmembrane domain (TMD0) of unknown function. In addition, although the vast majority of ABC proteins are active transporters, there are exceptions where evolution has adapted the ATPswitch mechanism, which appears to have evolved to drive transport to other activities. Thus, one mammalian ABC protein is an ion channel (CFTR; ABCC7) and another is a regulator of ion channel activity (SUR; ABCC8 and ABCC9). Other ABC proteins couple ATP binding and hydrolysis to the control of translation or DNA repair. Those that are not transporters are not covered in this special issue. We would like to thank the ABC community for devoting their time and energy to writing these chapters; we would also like to thank those who contributed to the field many stimulating thoughts, ideas, and good scientific times, though not specifically in writing . Finally, we thank Heini Murer from Pfluger’s Archives for his continuous support in making this special issue possible.

Prognostic Impact of Anti-Apoptotic and Multidrug Resistance Phenotypes in Elderly Acute Myeloid Leukemia Patients.

Elderly patients with acute myeloid leukemia (AML) have an unfavorable outcome, which has been related to both the poor performance status of many of these patients and the biological characteristics of the malignant clone, including the expression of multidrug resistance (MDR) phenotypes. We have quantitatively analyzed by flow cytometry the expression of apoptosis- (bcl-2, bax, APO2.7) and MDR- (P-gp, MRP, LRP) associated proteins in a group of 117 elderly (>65 years) uniformly treated de novo AML patients according to the Pethema LMA-98 protocol. Upon analyzing factors influencing the response to induction therapy, as expected, patients with good and intermediate cytogenetics (n=76) more frequently achieved morphological complete remission (mCR) (63%) than patients having poor cytogenetics (n=23) (mCR: 30%). In addition, CD34 expression also influenced response; thus, 81% (21/26) of CD34-negative patients achieved mCR vs only 46% (42/91) of CD34-positive cases (p=0,008). Neither age nor WBC counts showed a significant influence in response rate. As far as apoptosis and MRD proteins is concerned, interestingly, responding cases showed a lower expression of the bcl-2, MRP and LRP proteins (bcl-2 RFI (relative fluorescence intensity) 10±5.5 vs 13±6, p=0.02; MRP RFI 1.9±0.8 vs 2.7±1.8, p=0.008; LRP RFI 6.2±4.9 vs 8.7±7.5, p=0.01 in responding vs non-responding patients). By contrast, expression of APO 2.7, Bax, and MDR-1 did not influenced response. Analysis of relapse free survival showed that only the number of cycles of chemotherapy required to achieve mCR had prognostic influence (p=0.008), with no significant influence for age, WBC counts, CD34 expression, or cytogenetics. In turn, a high percentage of early apoptotic cells in bone marrow at diagnosis (p=0.01), as well as a low bcl-2/bax ratio (p=0.05), and low MRP expression (p=0.04) were associated with a prolonged RFS. Moreover, upon grouping AML patients according to the expression of MRP and the bcl-2/bax ratio (> and < of the mean of both parameters), only patients with both low MRP expression and low bcl-2/bax ratio (n=16) achieved a plateau phase in the RFS curve after 20 months of follow-up, while the remaining patients showed a continuous relapse trend.In summary, our results show that, in addition to high bcl-2 and bcl-2/bax ratio, a high expression of the LRP and MRP multidrug resistant proteins have an adverse prognostic influence in elderly AML patients. The combination of these parameters contribute to identify distinct groups of patients at a different risk of relapse.

Anticancer Multidrug Resistance Mediated by MRP1: Recent Advances in the Discovery of Reversal Agents

AbstractFor Abstract see ChemInform Abstract in Full Text.

The Mrp system: a giant among monovalent cation/proton antiporters?

Mrp systems are a novel and broadly distributed type of monovalent cation/proton antiporter of bacteria and archaea. Monovalent cation/proton antiporters are membrane transport proteins that catalyze efflux of cytoplasmic sodium, potassium or lithium ions in exchange for external hydrogen ions (protons). Other known monovalent cation antiporters are single gene products, whereas Mrp systems have been proposed to function as hetero-oligomers. A mrp operon typically has six or seven genes encoding hydrophobic proteins all of which are required for optimal Mrp-dependent sodium-resistance. There is little sequence similarity of Mrp proteins to other antiporters but three of these proteins have significant sequence similarity to membrane embedded subunits of ion-translocating electron transport complexes. Mrp antiporters have essential roles in the physiology of alkaliphilic and neutralophilic Bacillus species, nitrogen-fixing Sinorhizobium meliloti and in the pathogen Staphylococcus aureus, although these bacteria contain multiple monovalent cation/proton antiporters. The wide distribution of Mrp systems leads to the anticipation of important roles in an even wider variety of pathogens, extremophiles and environmentally important organisms. Here, the distribution, established physiological roles and catalytic activities of Mrp systems are reviewed, hypotheses regarding their complexity are discussed and major open questions about their function are highlighted.

Anticancer multidrug resistance mediated by MRP1: Recent advances in the discovery of reversal agents

Multidrug resistance protein 1 (MRP1) belongs to the ATP-binding cassette (ABC) transporter family. It is able to transport a broad range of anticancer drugs through cellular membranes, thus limiting their antiproliferative action. Since its discovery in 1992, MRP1 has been the most studied among MRP proteins, which now count nine members. Besides the biological work, which targets structure elucidation, binding sites location, and mode of action, most efforts have been focused on finding molecules which act as MRP1 inhibitors. In this review, we attempt to summarize and highlight studies dealing with modulators of MRP1-mediated multidrug resistance (MDR), which have been accomplished in the last 5 years. The reported MRP1 inhibitors are discussed according to their chemical class. Finally, we try to bring information on structure-activity relationship (SAR) aspects and how modulators might interact with MRP1. This study may facilitate the rational design of future modulators of MDR.

Expression and localization of human multidrug resistance protein (ABCC) family members in pancreatic carcinoma

Pancreatic ductal adenocarcinoma is among the top 10 causes of death from cancer in industrialized countries. In comparison with other gastrointestinal malignancies, pancreatic cancer is one of the tumors most resistant to chemotherapy. An important mechanism of tumor multidrug resistance is increased drug efflux mediated by several transporters of the ABC superfamily. Especially BCRP (ABCG2), MDR1 P-glycoprotein (ABCB1) and members of the MRP (ABCC) family are important in mediating drug resistance. The MRP family consists of 9 members (MRP1-MRP9) with MRP1-MRP6 being best characterized with respect to protein localization and substrate selectivity. Here, we quantified the mRNA expression of BCRP and of all MRP family members in normal human pancreas and pancreatic carcinoma and analyzed the mRNA level of the transporters most abundantly expressed in pancreatic tissue, BCRP, MRP1, MRP3, MRP4 and MRP5, in 37 tissue samples. In addition, we determined the localization of the 4 MRP proteins in normal human pancreas and in pancreatic carcinoma. The expression of BCRP, MRP1 and MRP4 mRNA did not correlate with tumor stage or grading. On the other hand, the expression of MRP3 mRNA was upregulated in pancreatic carcinoma samples and was correlated with tumor grading. The MRP5 mRNA level was significantly higher in pancreatic carcinoma tissue compared to normal pancreatic tissue. These data suggest that MRP3 and MRP5 are involved in drug resistance of pancreatic tumors and that quantitative analysis of their expression may contribute to predict the benefit of chemotherapy in patients with pancreatic cancer.

Substrates and inhibitors of efflux proteins interfere with the MTT assay in cells and may lead to underestimation of drug toxicity.

The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay is a widely used method in assessment of cytotoxicity and cell viability, and also in anti-cancer drug studies with tumour cells. These cells often express efflux proteins, such as P-glycoprotein (MDR1) or multidrug resistance (MDR) protein 1 (MRP1). MDCKII cells that overexpress these proteins (MDCKII-MDR1 or MDCKII-MRP1) and normal cells (MDCKII-wt) were used to investigate the effects of efflux pump activity on the results of MTT assay. Efflux protein activity was confirmed with calcein-AM efflux assay, and MTT assay was compared to another cytotoxicity test, the LDH release assay. Inhibition of MRP and MDR1 efflux proteins in MDCKII cell lines was associated paradoxically with increased reduction of MTT, implying an apparent increase in cell viability. This effect was seen when MK 571 (MRP1 and MRP2 inhibitor) or verapamil (MRP1 and MDR1 inhibitor) were used to block efflux protein activity. The calcein-AM efflux assay also showed that the MTT reagent inhibits the function of MDR1 in the MDCKII-MDR1 cell line. This study shows that MDR1 and possibly MRP proteins interfere with the MTT assay. Due to wide substrate specificity of efflux proteins and popularity of the MTT assay this interference is not trivial. Presence of any efflux protein substrate may therefore lead to underestimated results in MTT assay, thereby causing potential bias and erroneous conclusions in cytotoxicity studies.

Expression and immunolocalization of the multidrug resistance proteins, MRP1–MRP6 (ABCC1–ABCC6), in human brain

Multidrug resistance proteins (MRPs, symbol ABCC) are membrane glycoproteins that mediate the ATP-dependent export of organic anions, including cytotoxic and antiviral drugs, from cells. To identify MRP family members possibly involved in the intrinsic resistance of human brain to cytotoxic and antiviral drugs, we analyzed the expression and localization of MRP1–MRP6 in rapidly frozen perilesional samples of several regions of adult human brain obtained during neurosurgery. Quantitative polymerase chain reaction analysisshowed expression of MRP1, MRP2, MRP3, MRP4, and MRP5 mRNA, whereas MRP6 mRNA was below detectability. However, immunofluorescence microscopy of cryosections from human brain showed no reactivity for the MRP2 or MRP3 proteins. The proteins MRP1, MRP4, and MRP5 were clearly localized by confocal laser scanning microscopy to the luminal side of brain capillary endothelial cells. The MRP4 and MRP5 proteins were also detected in astrocytes of the subcortical white matter. Notably, MRP5 protein was present in pyramidal neurons. MRP proteins may, thus, contribute to the cellular efflux of endogenous anionic glutathione or glucuronate conjugates (substrates for MRP1), cyclic nucleotides (substrates for MRP4 and MRP5), or glutathione (co-substrate for MRP1 and MRP4); in addition, they may play an important role in the resistance of the brain to several cytotoxic and antiviral drugs.

Increased perivascular synovial membrane expression of myeloid-related proteins in psoriatic arthritis.

To analyze S-100 protein expression, in the form of myeloid-related protein 8 (MRP8), MRP14, and the heterodimer MRP8/MRP14, in psoriatic arthritis (PsA) patients compared with rheumatoid arthritis (RA) and spondylarthropathy (SpA) patients, and to determine the effect of methotrexate (MTX) on the MRP antigen expression in PsA patients. Serum, synovial fluid (SF), and synovium (taken at arthroscopy) samples were obtained from PsA (before and after MTX treatment), RA, and SpA patients. Concentrations of MRP8/MRP14 in serum and SF were measured by enzyme-linked immunosorbent assay. Expression of MRP8, MRP14, and MRP8/MRP14 in synovium was determined by immunohistochemistry. MRP8, MRP14, and MRP8/MRP14 levels were increased in serum, SF, and synovium from PsA, RA, and SpA patients. In all 3 groups, paired samples of serum and SF showed significantly higher MRP8/MRP14 levels in SF (mean +/- SD 15310 +/- 16999 ng/ml [median 11400]) than in serum (908 +/- 679 ng/ml [median 695]) (P = 0.0001). MRP8/MRP14 levels in serum correlated with systemic parameters of disease activity (erythrocyte sedimentation rate [ESR] r = 0.55, P = 0.005; C-reactive protein [CRP] level r = 0.55, P = 0.005), whereas levels in SF correlated with local parameters of disease activity (white blood cell count r = 0.45, P = 0.01; acute-phase serum amyloid A level r = 0.32, P = 0.03). MRP expression was significantly higher in the synovial sublining layer (SLL) of PsA patients compared with RA and SpA patients. MRP antigens were predominantly expressed in perivascular areas of the SLL in PsA patients. Following MTX treatment, MRP expression in serum and synovium from PsA patients was significantly reduced. Serum levels of MRP were more sensitive to the effects of MTX than were the ESR, CRP, or clinical joint scores. MRP levels in serum and SF correlate with local and systemic inflammation and are equally increased in PsA, RA, and SpA patients. In contrast, MRP8, MRP14, and MRP8/MRP14 expression in the SLL of PsA patients is increased, particularly in perivascular regions, compared with that in RA and SpA patients, suggesting a central role of MRP proteins in transendothelial migration of leukocytes in PsA. Moreover, MRP expression is reduced following MTX treatment. MRP proteins may represent a novel therapeutic target in inflammatory arthritis.

In vitro and in vivo hepatic transport of the magnetic resonance imaging contrast agent B22956/1: role of MRP proteins

The molecular mechanisms of the hepatic transport of B22956/1, a new gadolinium complex from the class of intravascular contrast agents for MRI, which undergoes extensive biliary elimination, were studied. Biliary and urinary elimination of B22956/1 were measured in normal and in mutant MRP2 lacking rats (TR −); cellular trafficking of the compound was assessed in wild and MRP1 or MRP2 transfected MDCKII cells. Eight hours after IV injection of B22956/1, 90±8% of the dose was recovered in the bile of normal rats. By contrast, in TR − rats, the biliary excretion was significantly lower (14±3%) while 55±9% of the compound was found in urine. In vitro, the cellular accumulation of B22956/1 was significantly lower in both MRP1 and MRP2 transfected cells as compared to wild type MDCKII cells, and the cellular efflux was prevented by the MRP inhibitor MK571, indicating the involvement of both MRP2 and MRP1 in the transport of B22956/1. Due to the distinct cellular localization of the proteins, MRP2 accounts for the biliary and urinary excretion of the compound, while MRP1 prevents cellular accumulation of the MRI agent. B22956/1 may be useful in clinical conditions where a defective biliary transport is present.

Genetic studies of mrp, a locus essential for cellular aggregation and sporulation of Myxococcus xanthus.

Under starvation conditions, Myxococcus xanthus undergoes a complex developmental process which includes cellular aggregation and sporulation. A transposon insertion mutant (the Tn5-Omega280 mutant) with defects in both aggregation and sporulation was analyzed in this study. The Tn5-Omega280 mutant was found to have a disrupted NtrC-like response regulator designated Myxococcus regulatory protein B (mrpB). Further sequencing analyses revealed a histidine kinase homolog (mrpA) immediately upstream of mrpB and a cyclic AMP receptor protein-like transcriptional regulator (mrpC) downstream of mrpB. In-frame deletion analyses revealed that both the mrpB and mrpC genes were required for cellular aggregation and sporulation but that only mrpA was required for sporulation only. Site-specific mutagenesis of the putative phosphorylation site of MrpB, D58, showed that a D58A mutation caused defects in both aggregation and sporulation but that a D58E mutation resulted in only a sporulation defect. Further genetic and molecular analyses with reporter genes and reverse transcription-PCR indicated that mrpA and mrpB are cotranscribed but that mrpC is transcribed independently and that all of these genes are developmentally regulated. In addition, MrpB is essential for transcription of mrpC and MrpC regulates its own transcription. These data indicate that Mrp proteins are important components required for M. xanthus development. The complicated interaction between Mrp proteins may play an important role in regulating developmental gene expression in M. xanthus.