Cytosolic and nuclear mitogen-activated protein kinases are regulated by distinct mechanisms

We have investigated the regulation and localization of mitogen-activated protein kinase (MAPK) and mitogen-activated protein kinase kinase (MAPKK) in both cytosolic and nuclear fractions of glomerular mesangial cells. p42 MAPK was localized by both immunoblot and kinase activity in both cytosol and nucleus and was rapidly activated, in both fractions, by fetal bovine serum and TPA. Downregulation of protein kinase C (PKC) by TPA inhibited stimulation of cytosolic p42 MAPK, but unexpectedly had no effect on stimulated p42 MAPK in the nucleus. Next we studied the upstream kinase p45 MAPKK by indirect immunofluorescence microscopy, Western blot analysis, and kinase specific activity. Unlike MAPK, p45 MAPKK is almost exclusively cytosolic in resting cells and kinase activity stimulated by TPA is restricted to the cytosol. Interestingly, PKC downregulation for 24 h with TPA dramatically enhanced nuclear MAPKK as assessed by all three techniques. Cytosolic stimulated MAPKK was attenuated in PKC downregulation. Collectively these results show that in mesangial cells: (i) p42 MAPK and p45 MAPKK localize in both the cytosol and the nucleus, and (ii) PKC exerts a negative effect on nuclear MAPKK activity as documented by PKC downregulation, which augments p45 MAPPK nuclear mass and activity. These results indicate that the dual regulation of these two kinases is under differential control in the cytosol and the nucleus.     

Dendritic cell survival and maturation are regulated by different signaling pathways

OBJECTIVE: Quantification of serum nucleotide pyrophosphohydrolase (NTPPHase) activity in healthy subjects and in patients with various rheumatic diseases or with quad/hemiplegia, hemodialysis, or renal transplant. METHODS: Colorimetric assay of enzyme activity in serum. RESULTS: Serum NTPPHase activity in 85 healthy subjects was independent of age or sex and was highly reproducible in each individual. The biologic and methodologic coefficients of variation were nearly identical. Elevated enzyme levels were found in sera from patients with osteoarthritis/spondylosis, calcium pyrophosphate dihydrate (CPPD) crystal deposition, scleroderma, fibromyalgia, or hemodialysis. Renal transplant patients receiving cyclosporine had the highest enzyme activity of any group, whereas transplant patients not taking this drug had normal levels. Histograms of values in all groups showed a normal distribution. CONCLUSION: Serum NTPPHase activity levels were significantly elevated in patients with degenerative arthritis whether or not CPPD crystals were present, in patients with either scleroderma or fibromyalgia, and in patients receiving hemodialysis therapy or taking cyclosporine.     

Two functional soybean genes encoding p34cdc2 protein kinases are regulated by different plant developmental pathways

We have isolated two cDNA clones (cdc2-S5 and cdc2-S6) encoding p34cdc2 protein kinases, homologs of yeast cdc2/CDC28 genes, from a soybean nodule cDNA library. The two sequences share 90% sequence homology in the coding regions. The 5' and 3' noncoding regions are distinct from each other, however, indicating that at least two genes encode p34cdc2 protein kinases in soybean. Both sequences can rescue the cdc28 mutation in Saccharomyces cerevisiae but rescue it with different efficiency. Genomic Southern analysis showed the existence of two copies for each of these genes, which are not closely linked and are nonallelic. The relative expression level of the two soybean p34cdc2 genes varies in different tissues. Expression of cdc2-S5 is higher in roots and root nodules, whereas cdc2-S6 is more actively expressed in aerial tissues, indicating that regulation of these two p34cdc2 genes is coupled with plant developmental pathways. Expression of cdc2-S5 is, furthermore, enhanced after Rhizobium infection, whereas cdc2-S6 fails to respond, suggesting that cdc2-S5 plays a role in nodule initiation and organogenesis. This latter gene preferentially responds to auxin (alpha-naphthaleneacetic acid) treatment, indicating that phytohormones may be involved in the control of cell division mediated by Rhizobium infection. Thus, different p34cdc2 protein kinases may control cell division in different tissues in a multicellular organism and respond to different signals--e.g., phytohormones.     

Stabilization and activation of p53 are regulated independently by different phosphorylation events

Rheumatoid factors (RF) recognize conformational determinants located within the Fc portion of IgG. By analyzing a panel of monoclonal rheumatoid arthritis (RA)-derived RFs, we previously demonstrated that the somatically generated light chain complementarity-determining region 3 (CDR3) contributes to RF specificity. We have now generated a panel of heavy chain mutants of the B'20 Ab, a high affinity RA-derived IgM RF. B'20 also binds avidly to protein A and weakly to ssDNA and tetanus toxoid. B9601, a RF negative Ab that is highly homologous to B'20 but does not bind any of the Ags tested, and RC1, a low affinity polyreactive RF, were used to generate heavy chain mutants with framework (FR) and CDR switches. The mutated heavy chains were cotransfected into a myeloma cell line with the germline counterpart of the B'20 light chain, and the expressed Ig tested for antigenic specificity. We show that both RF specificity and polyreactivity of B'20 is dependent on its unique heavy chain CDR3 region. Replacement with a B9601 CDR3 shortened to the same length as the B'20 CDR3, and with only 5 amino acid differences, did not restore Fc binding. Conversely, absence of protein A binding of B9601 is due to the presence of a serine residue at position 82a in the B9601 heavy chain FR3 region. Together, our data suggest that Ig gene recombination events can generate B cells with autoantibody specificities in the preimmune repertoire. Abnormal release, activation, expansion, or mutation of such cells might all contribute to the generation of a high titer RF response in patients with RA.     

Amino acid-amino acid contacts at the cooperativity interface of the bacteriophage lambda and P22 repressors

The bacteriophage lambda repressor and its relatives bind cooperatively to adjacent as well as artificially separated operator sites. This cooperativity is mediated by a protein-protein interaction between the DNA-bound dimers. Here we use a genetic approach to identify two pairs of amino acids that interact at the dimer-dimer interface. One of these pairs is nonconserved in the aligned sequences of the lambda and P22 repressors; we show that a lambda repressor variant bearing the P22 residues at these two positions interacts specifically with the P22 repressor. The other pair consists of a conserved ion pair; we reverse the charges at these two positions and demonstrate that, whereas the individual substitutions abolish the interaction of the DNA-bound dimers, these changes in combination restore the interaction of both lambdacI and P22c2 dimers.     

Leptin and UCP1 genes are reciprocally regulated in brown adipose tissue

In a previous work we showed that only unilocular brown adipocytes express leptin. In order to investigate the relationship between leptin gene expression, brown adipocyte activity (UCP1) and morphology, we studied brown adipose tissues of mice (C57BL, female, 7 weeks old) acclimated at different temperatures (19 degrees C and 28 degrees C). Northern blot analysis revealed higher leptin and lower UCP1 mRNA levels in mice exposed to 28 degrees C than in the group acclimated at 19 degrees C. Also protein expression (immunohistochemistry) differed in the two groups: at 28 degrees C brown adipocytes were positive for leptin and only weakly positive for UCP1, while at 19 degrees C they were leptin-negative and UCP1-positive. In the former group the morphology was mainly unilocular. Our data suggest that in brown adipocytes of warm-acclimated mice leptin expression is closely related to their hypoactive functional stage, as evidenced by their low level of UCP1 synthesis and the morphological rearrangement of the lipid content (unilocularity).     

The affinities of procolipase and colipase for interfaces are regulated by lipids

It has been suggested that at physiological pH, the trypsin-catalyzed activation of the lipase cofactor, procolipase, to colipase has no consequence for intestinal lipolysis and serves primarily to release the N-terminal pentapeptide, enterostatin, a satiety factor (Larsson, A., and C. Erlanson-Albertsson 1991. The effect of pancreatic procolipase and colipase on pancreatic lipase activation. Biochim. Biophys. Acta 1083:283-288). This hypothesis was tested by measuring the adsorption of [14C]colipase to monolayers of 1-stearoyl-2-oleoyl-sn-3-glycerophosphocholine and 13, 16-cis, cis-docosadienoic acid in the presence and absence of procolipase. With saturating [14C]colipase in the subphase, the surface excess of [14C]colipase is 29% higher than that of procolipase, indicating that colipase packs more tightly in the interface. With [14C]colipase-procolipase mixtures, the proteins compete equally for occupancy of the argon-buffer interface. However, if a monolayer of either or both lipids is present, [14C]colipase dominates the adsorption process, even if bile salt is present in the subphase. If [14C]colipase and procolipase are premixed for > 12 h at pH approximately 8, this dominance is partial. If they are not premixed, procolipase is essentially excluded from the interface, even if procolipase is added before [14C]colipase. These results suggest that the tryptic cleavage of the N-terminal pentapeptide of procolipase may be of physiological consequence in the intestine.     

Molecular mechanisms and selection influence the generation of the human V lambda J lambda repertoire

To define the lambda light chain repertoire in humans, a single-cell PCR technique using genomic DNA obtained from individual peripheral B cells was employed. Of the 30 known functional V lambda genes, 23 were detected in either the nonproductive or productive repertoires. Specific V lambda genes, including 2A2, 2B2, 1G, and 4B, were overexpressed in the nonproductive repertoire, whereas some V lambda genes, such as 3R, 2A2, 2B2, 1C, 1G, and 1B, were overexpressed in the productive repertoire. Comparison of the nonproductive and productive repertoires indicated that no V lambda genes were positively selected, whereas a number of V lambda genes, including 4C, 1G, 5B, and 4B, were negatively regulated. All four of the functional J lambda segments were found in both repertoires, with J lambda 7 observed most often. Evidence of terminal deoxynucleotidyltransferase activity was noted in nearly 80% of nonproductive V lambda J lambda rearrangements, and exonuclease activity was apparent in the majority. Despite this, the mean CDR3 length was 30 base pairs in both productive and nonproductive repertoires, suggesting that it was tightly regulated at the molecular level. These results have provided new insights into the dimensions of the human V lambda repertoire and the influences that shape it.     

Gelsolin and functionally similar actin-binding proteins are regulated by lysophosphatidic acid

An extensive survey was carried out for compounds capable of regulating actin-binding proteins in a manner similar to phosphatidylinositol 4,5 bisphosphate (PI 4,5-P2). For this purpose we developed a sensitive assay involving release of radioactively phosphorylated actin from the fragminP-actin complex. We found that the structurally simplest lysophospholipid, lysophosphatidic acid (LPA), dissociated the complex between fragminP and actin, whereas other lysophospholipids or sphingosine-1-phosphate were inactive. Furthermore, LPA inhibited the F-actin severing activity of human gelsolin, purified from plasma or as recombinant protein, mouse adseverin and Physarum fragminP. Dissociation of actin-containing complexes by LPA analyzed by gelfiltration indicated that LPA is active as a monomer, in contrast to PI 4,5-P2. We further show that binding of LPA to these actin-regulatory proteins promotes their phosphorylation by pp60(c-src). A PI 4,5-P2-binding peptide counteracted the effects mediated by LPA, suggesting that LPA binds to the same target region in these actin-binding proteins. When both LPA and PI 4,5-P2 were used in combination we found that LPA reduced the threshold concentration at which PI 4,5-P2 was active. Significantly, LPA promoted the release of gelsolin from barbed actin filaments in octylglucoside-permeabilized human platelets. These results suggest that lysophosphatidic acid could act as an intracellular modulator of actin-binding proteins. Our findings can also explain agonist-induced changes in the actin cytoskeleton that are not mediated by polyphosphoinositides.     

Expression of putative fatty acid transporter genes are regulated by peroxisome proliferator-activated receptor alpha and gamma activators in a tissue- and inducer-specific manner

Regulation of gene expression of three putative long-chain fatty acid transport proteins, fatty acid translocase (FAT), mitochondrial aspartate aminotransferase (mAspAT), and fatty acid transport protein (FATP), by drugs that activate peroxisome proliferator-activated receptor (PPAR) alpha and gamma were studied using normal and obese mice and rat hepatoma cells. FAT mRNA was induced in liver and intestine of normal mice and in hepatoma cells to various extents only by PPARalpha-activating drugs. FATP mRNA was similarly induced in liver, but to a lesser extent in intestine. The induction time course in the liver was slower for FAT and FATP mRNA than that of an mRNA encoding a peroxisomal enzyme. An obligatory role of PPARalpha in hepatic FAT and FATP induction was demonstrated, since an increase in these mRNAs was not observed in PPARalpha-null mice. Levels of mAspAT mRNA were higher in liver and intestine of mice treated with peroxisome proliferators, while levels in hepatoma cells were similar regardless of treatment. In white adipose tissue of KKAy obese mice, thiazolidinedione PPARgamma activators (pioglitazone and troglitazone) induced FAT and FATP more efficiently than the PPARalpha activator, clofibrate. This effect was absent in brown adipose tissue. Under the same conditions, levels of mAspAT mRNA did not change significantly in these tissues. In conclusion, tissue-specific expression of FAT and FATP genes involves both PPARalpha and -gamma. Our data suggest that among the three putative long-chain fatty acid transporters, FAT and FATP appear to have physiological roles. Thus, peroxisome proliferators not only influence the metabolism of intracellular fatty acids but also cellular uptake, which is likely to be an important regulatory step in lipid homeostasis.     

Dendritic cell chemotaxis and transendothelial migration are induced by distinct chemokines and are regulated on maturation

The capacity of dendritic cells (DC) to initiate immune responses is dependent on their specialized migratory and tissue homing properties. Chemotaxis and transendothelial migration (TEM) of DC were studied in vitro. Immature DC were generated by culture of human monocytes in granulocyte-macrophage colony-stimulating factor and IL-4. These cells exhibited potent chemotaxis and TEM responses to the CC chemokines macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, RANTES, and monocyte chemotactic protein-3, and weak responses to the CC chemokine MIP-3beta and the CXC chemokine stromal cell-derived factor (SDF)-1alpha. Maturation of DC induced by culture in lipopolysaccharide, TNF-alpha or IL-1beta reduced or abolished responses to the former CC chemokines but markedly enhanced responses to MIP-3beta and SDF-1alpha. This correlated with changes in chemokine receptor expression: CCR5 expression was reduced while CXCR4 expression was enhanced. These findings suggest two stages for regulation of DC migration in which one set of chemokines may regulate recruitment into or within tissues, and another egress from the tissues.     

Isolation of developmentally regulated genes by differential display screening of cDNA libraries

mRNA differential display RT-PCR has been extensively used for the isolation of genes differentially expressed between RNA populations. We have assessed its utility for the identification of developmentally regulated genes in plasmid cDNA libraries derived from individual tissues dissected from early mouse embryos. Using plasmid Southern blot hybridisation as a secondary screen, we are able to identify such genes and show by whole-mount in situ hybridisation that their expression pattern is that expected from the differential display profile.