Lee A. Weber

A Role for p38MAPK/HSP27 Pathway in Smooth Muscle Cell Migration

Smooth muscle cells are exposed to growth factors and cytokines that contribute to pathological states including airway hyperresponsiveness, atherosclerosis, angiogenesis, smooth muscle hypertrophy, and hyperplasia. A common feature of several of these conditions is migration of smooth muscle beyond the initial boundary of the organ. Signal transduction pathways activated by extracellular signals that instigate migration are mostly undefined in smooth muscles. We measured migration of cultured tracheal myocytes in response to platelet-derived growth factor, interleukin-1β, and transforming growth factor-β. Cellular migration was blocked by SB203580, an inhibitor of p38MAPK. Time course experiments demonstrated increased phosphorylation of p38MAPK. Activation of p38MAPK resulted in the phosphorylation of HSP27 (heat shockprotein 27), which may modulate F-actin polymerization. Inhibition of p38MAPK activity inhibited phosphorylation of HSP27. Adenovirus-mediated expression of activated mutant MAPK kinase 6b(E), an upstream activator for p38MAPK, increased cell migration, whereas overexpression of p38α MAPK dominant negative mutant and an HSP27 phosphorylation mutant blocked cell migration completely. The results indicate that activation of the p38MAPK pathway by growth factors and proinflammatory cytokines regulates smooth muscle cell migration and may contribute to pathological states involving smooth muscle dysfunction.

In Vivo Chaperone Activity of Heat Shock Protein 70 and Thermotolerance

ABSTRACT Heat shock protein 70 (Hsp70) is thought to play a critical role in the thermotolerance of mammalian cells, presumably due to its chaperone activity. We examined the chaperone activity and cellular heat resistance of a clonal cell line in which overexpression of Hsp70 was transiently induced by means of the tetracycline-regulated gene expression system. This single-cell-line approach circumvents problems associated with clonal variation and indirect effects resulting from constitutive overexpression of Hsp70. The in vivo chaperone function of Hsp70 was quantitatively investigated by using firefly luciferase as a reporter protein. Chaperone activity was found to strictly correlate to the level of Hsp70 expression. In addition, we observed an Hsp70 concentration dependent increase in the cellular heat resistance. In order to study the contribution of the Hsp70 chaperone activity, heat resistance of cells that expressed tetracycline-regulated Hsp70 was compared to thermotolerant cells expressing the same level of Hsp70 plus all of the other heat shock proteins. Overexpression of Hsp70 alone was sufficient to induce a similar recovery of cytoplasmic luciferase activity, as does expression of all Hsps in thermotolerant cells. However, when the luciferase reporter protein was directed to the nucleus, expression of Hsp70 alone was not sufficient to yield the level of recovery observed in thermotolerant cells. In addition, cells expressing the same level of Hsp70 found in heat-induced thermotolerant cells containing additional Hsps showed increased resistance to thermal killing but were more sensitive than thermotolerant cells. These results suggest that the inducible form of Hsp70 contributes to the stress-tolerant state by increasing the chaperone activity in the cytoplasm. However, its expression alone is apparently insufficient for protection of other subcellular compartments to yield clonal heat resistance to the level observed in thermotolerant cells.

Changes in the stability of a human H3 histone mRNA during the HeLa cell cycle.

A major component of the regulation of histone protein synthesis during the cell cycle is the modulation of the half-life of histone mRNA. We have uncoupled transcriptional and posttranscriptional regulation by using a Drosophila hsp70-human H3 histone fusion gene that produces a marked human H3 histone mRNA upon heat induction. Transcription of this gene can be switched on and off by raising and lowering cell culture temperatures, respectively. HeLa cell lines containing stably integrated copies of the fusion gene were synchronized by double thymidine block. Distinct populations of H3 histone mRNA were produced by heat induction in early S-phase, late S-phase, or G2-phase cells, and the stability of the induced H3 histone mRNA was measured. The H3 histone mRNA induced during early S phase decayed with a half-life of 110 min, whereas the same transcript induced during late S phase had a half-life of 10 to 15 min. The H3 histone mRNA induced in non-S-phase cells is more stable than that induced in late S phase, with a half-life of 40 min. Thus, the stability of histone mRNA is actively regulated throughout the cell cycle. Our results are consistent with an autoregulatory model in which the stability of histone mRNA is determined by the level of free histone protein in the cytoplasm.

Stress protection by a fluorescent Hsp27 chimera that is independent of nuclear translocation or multimeric dissociation

Abstract A chimeric protein consisting of enhanced green fluorescent protein (EGFP) fused to the N-terminus of human Hsp27 conferred stress protection in human A549 lung carcinoma and murine L929 cells that were stably transfected to express the chimera constitutively. The resultant protection was comparable with that in the same cell lines when they were transfected to express corresponding levels of Hsp27. Unlike L929 cells, A549 cells exhibit endogenous Hsp27 expression, whose expression was inhibited in proportion to the amount of fluorescent chimera expressed, suggesting that the A549 cells recognized the latter as Hsp27. Upregulation of Hsp27 or chimeric Hsp27 in all transfected cell lines (stable or transient transfection) caused no measurable change in cellular glutathione levels, indicating that glutathione played no role in the stress protection associated with either protein. Chimeric Hsp27 had a monomeric molecular weight of 55 kDa (that of Hsp27 plus EGFP) in both cell types and formed a 16-mer complex twice as massive as that formed by Hsp27. Heat shock or sodium arsenite induced phosphorylation of both chimeric Hsp27 and Hsp27, which resulted in the disaggregation of Hsp27 multimers in both cell types and disaggregation of 20% of the chimeric multimers in L929 cells. But chimeric Hsp27 multimers did not disaggregate after stress in A549 cells. Epifluorescence and confocal microscopy demonstrated that chimeric Hsp27 was restricted to the cytoplasm under normal growth conditions and after heat shock in all cells. This study supports the conclusions that Hsp27 stress protection requires neither its translocation into the nucleus nor the dissociation of its multimeric complex. Furthermore, it demonstrates that fluorescent chimeras of heat shock proteins can be functional and used to observe the proteins distribution within living cells.

Cloning and sequencing of a cDNA encoding the canine HSP27 protein

The nucleotide (nt) sequence encoding a 27-kDa heat-shock protein (HSP27) was determined from cDNAs cloned from a canine smooth muscle library. The primary structure deduced from the nt sequence reveals a 209-amino-acid protein having 86-89% identity with human, mouse, rat and hamster small HSP. Similar to human HSP27, the canine protein contains three Ser residues that are potential MAPKAP kinase II substrates.

Intraspecific variation in the rainbow trout mitochondrial DNA genome.

Four complete mitochondrial DNA genomes for rainbow trout, Oncorhynchus mykiss, were sequenced and compared to the previously reported complete mitochondrial sequence. Analyses revealed 13 additional bp and 31 insertion/deletion (indels) sites between the four new sequences and the previously published sequence. Twenty-eight indels were single base pairs while the remaining were multiple base pair insertions, two in the ATPase6 gene, one of three bases and one of nine bases, and a two base insert in the control region. Indels in the new genomes were compared to a wide range of salmonid species and corresponded with sequences in closely related species, indicating that errors were likely incorporated into the previously published sequence. Although variable nucleotide positions were observed throughout the newly sequenced genomes, non-synonymous amino acid substitutions were observed in only six of seven ND genes. While there were only 14 amino acid substitutions between genomes, K(a)/K(s) ratios ranged between 0 and 1. A single amino acid deletion in the ND1 coding region unique to rainbow trout was also detected. Using complete mitochondrial genomes for all sequenced Salmonidae species, a phylogenetic analysis was performed to establish a salmonid mitochondrial phylogeny revealing support for Salvelinus, rather than Salmo, as the sister taxa to Oncorhynchus.