Ionizing radiation induces myofibroblast differentiation via lactate dehydrogenase.

Pulmonary fibrosis is a common and dose-limiting side-effect of ionizing radiation used to treat cancers of the thoracic region. Few effective therapies are available for this disease. Pulmonary fibrosis is characterized by an accumulation of myofibroblasts and excess deposition of extracellular matrix proteins. Although prior studies have reported that ionizing radiation induces fibroblast to myofibroblast differentiation and collagen production, the mechanism remains unclear. Transforming growth factor-ß (TGF-ß) is a key profibrotic cytokine that drives myofibroblast differentiation and extracellular matrix production. However, its activation and precise role in radiation-induced fibrosis are poorly understood. Recently, we reported that lactate activates latent TGF-ß through a pH-dependent mechanism. Here, we wanted to test the hypothesis that ionizing radiation leads to excessive lactate production via expression of the enzyme lactate dehydrogenase-A (LDHA) to promote myofibroblast differentiation. We found that LDHA expression is increased in human and animal lung tissue exposed to ionizing radiation. We demonstrate that ionizing radiation induces LDHA, lactate production, and extracellular acidification in primary human lung fibroblasts in a dose-dependent manner. We also demonstrate that genetic and pharmacologic inhibition of LDHA protects against radiation-induced myofibroblast differentiation. Furthermore, LDHA inhibition protects from radiation-induced activation of TGF-ß. We propose a profibrotic feed forward loop, in which radiation induces LDHA expression and lactate production, which can lead to further activation of TGF-ß to drive the fibrotic process. These studies support the concept of LDHA as an important therapeutic target in radiation-induced pulmonary fibrosis.

Role of CXCR2 in cigarette smoke-induced lung inflammation.

It has been hypothesized that the destruction of lung tissue observed in smokers with chronic obstructive pulmonary disease and emphysema is mediated by neutrophils recruited to the lungs by smoke exposure. This study investigated the role of the chemokine receptor CXCR2 in mediating neutrophilic inflammation in the lungs of mice acutely exposed to cigarette smoke. Exposure to dilute mainstream cigarette smoke for 1 h, twice per day for 3 days, induced acute inflammation in the lungs of C57BL/6 mice, with increased neutrophils and the neutrophil chemotactic CXC chemokines macrophage inflammatory protein (MIP)-2 and KC. Treatment with SCH-N, an orally active small molecule inhibitor of CXCR2, reduced the influx of neutrophils into the bronchoalveolar lavage (BAL) fluid. Histological changes were seen, with drug treatment reducing perivascular inflammation and the number of tissue neutrophils. beta-Glucuronidase activity was reduced in the BAL fluid of mice treated with SCH-N, indicating that the reduction in neutrophils was associated with a reduction in tissue damaging enzymes. Interestingly, whereas MIP-2 and KC were significantly elevated in the BAL fluid of smoke exposed mice, they were further elevated in mice exposed to smoke and treated with drug. The increase in MIP-2 and KC with drug treatment may be due to the decrease in lung neutrophils that either are not present to bind these chemokines or fail to provide a feedback signal to other cells producing these chemokines. Overall, these results demonstrate that inhibiting CXCR2 reduces neutrophilic inflammation and associated lung tissue damage due to acute cigarette smoke exposure.

Increasing the frequency of T-cell precursors specific for a cryptic epitope of hen-egg lysozyme converts it to an immunodominant epitope.

Efforts to understand the mechanisms that govern how immunodominant T-cell epitopes are selected from protein antigens have focused mostly on differences in the efficiency of processing and presentation of peptide/major histocompatibility complex (MHC) complexes by antigen-presenting cells, while little attention has been directed at the role of the T-cell repertoire. In this report, the influence of the T-cell repertoire on immunodominance was investigated using transgenic mice that express the beta chain from a T-cell receptor specific for a cryptic Ek restricted epitope of hen-egg lysozyme, HEL85-96. In these mice, the frequency of HEL85-96-specific T-cell precursors is increased 10-20-fold over non-transgenic mice. Transgenic mice respond as well as non-transgenic controls to intact HEL, even though they respond poorly or not at all to a variety of other antigens, including the dominant H-2k restricted epitopes of HEL. Following immunization with native HEL, the only HEL peptide that could recall a response in vitro in the transgenic mice was HEL85-96. Therefore, this normally cryptic epitope is the sole immunodominant epitope in the transgenic mice, and this alteration in immune response is due solely to an increase in the frequency of specific T-cell precursors. An analysis of four additional H-2k restricted cryptic epitopes of HEL suggests that three are similarly limited by T-cell frequency, and that only one is consistent with a defect in efficient antigen presentation. This indicates that there are at least two different types of cryptic epitopes, one in which crypticity is caused by inefficient processing or presentation, and another in which the frequency of specific T-cell progenitors is limiting.

Electroporation-mediated replacement of a positively and negatively selectable beta-tubulin gene in Tetrahymena thermophila.

Replacement of lysine-350 by methionine in the beta-tubulin gene of Chlamydomonas confers resistance to microtubule-depolymerizing drugs and increased sensitivity to the microtubule-stabilizing drug taxol. This mutation was created in cloned BTU1, one of two coexpressed beta-tublin genes of Tetrahymena thermophila. When introduced by electroporation, the mutated gene transformed Tetrahymena exclusively by gene replacement at the homologous locus. Taxol-sensitive transformants could be retransformed with a wild-type gene and selection for taxol resistance. Analyses of phenotypic assortment and of the mRNA in transformed cells suggest that complete replacement of the BTU1 gene in the polyploid macronucleus can be obtained. These studies demonstrate the utility of this marker for studying tublin gene function and show that electroporation allows facile gene replacement in Tetrahymena.

Independent evolutionary origin of histone H3.3-like variants of animals and Tetrahymena.

All three genes encoding histone H3 proteins were cloned and sequenced from Tetrahymena thermophila. Two of these genes encode a major H3 protein identical to that of T. pyriformis and 87% identical to the major H3 of vertebrates. The third gene encodes hv2, a quantitatively minor replication independent (replacement) variant. The sequence of hv2 is only 85% identical to the animal replacement variant H3.3 and is the most divergent H3 replacement variant described. Phylogenetic analysis of 73 H3 protein sequences suggests that hv2, H3.3, and the plant replacement variant H3.III evolved independently, and that H3.3 is not the ancestral H3 gene, as was previously suggested (Wells, D., Bains, W., and Kedes, L. 1986, J. Mol. Evol., 23: 224-241). These results suggest it is the replication independence and not the particular protein sequence that is important in the function of H3 replacement variants.

Phylogenetic analysis of the core histones H2A, H2B, H3, and H4.

Despite the ubiquity of histones in eukaryotes and their important role in determining the structure and function of chromatin, no detailed studies of the evolution of the histones have been reported. We have constructed phylogenetic trees for the core histones H2A, H2B, H3, and H4. Histones which form dimers (H2A/H2B and H3/H4) have very similar trees and appear to have co-evolved, with the exception of the divergent sea urchin testis H2Bs, for which no corresponding divergent H2As have been identified. The trees for H2A and H2B also support the theory that animals and fungi have a common ancestor. H3 and H4 are 10-fold less divergent than H2A and H2B. Three evolutionary histories are observed for histone variants. H2A.F/Z-type variants arose once early in evolution, while H2A.X variants arose separately, during the evolution of multicellular animals. H3.3-type variants have arisen in multiple independent events.

Four distinct and unusual linker proteins in a mitotically dividing nucleus are derived from a 71-kilodalton polyprotein, lack p34cdc2 sites, and contain protein kinase A sites.

Tetrahymena thermophila micronuclei contain four linker-associated proteins, alpha, beta, gamma, and delta. Synthetic oligonucleotides based on N-terminal protein sequences of beta and gamma were used to clone the micronuclear linker histone (MLH) gene. The MLH gene is single copy and is transcribed into a 2.4-kb message encoding all four linker-associated proteins. The message is translated into a polypeptide (Mic LH) that is processed at the sequence decreases RTK to give proteins whose amino acid sequences differ markedly from each other, from the sequence of macronuclear H1, and from sequences of typical H1s of other organisms. This represents the first example of multiple chromatin proteins derived from a single polyprotein. The delta protein consists largely of two high-mobility-group (HMG) boxes. An evolutionary analysis of HMG boxes indicates that the delta HMG boxes are similar to the HMG boxes of tsHMG, a protein that appears in elongating mouse spermatids when they condense and cease transcription, suggesting that delta could play a similar role in the micronucleus. The micronucleus divides mitotically, while the macronucleus divides amitotically. Surprisingly, macronuclear H1 but not Mic LH contains sequences resembling p34cdc2 kinase phosphorylation sites, while each of the Mic LH-derived proteins contains a typical protein kinase A phosphorylation site in its carboxy terminus.

A temperature-sensitive cell cycle arrest mutation affecting H1 phosphorylation and nuclear localization of a small heat shock protein in Tetrahymena thermophila.

This report describes a temperature-sensitive Tetrahymena thermophila cell cycle arrest mutant that is also deficient in its heat shock response. Mutants incubated at 41 degrees C undergo rapid dephosphorylation of macronuclear histone H1, in contrast to wild-type cells which hyperphosphorylate H1 under the same conditions. Dephosphorylation is specific to H1 and is associated with a threefold decrease in the level of H1 kinase activity in macronuclei isolated from heat-shocked mutants. A small nuclear heat shock protein, sp29c, is synthesized and phosphorylated normally in the mutant cells but fails to accumulate in macronuclei. Nuclear transport of other heat shock proteins is unaffected. Mutant cells die slowly at 41 degrees C, a temperature at which wild-type cells resume normal growth after a brief lag. Wild-type cells acquire thermotolerance (competence to survive a 3-h heat shock at 43 degrees C) after a 1-h treatment at 41 degrees C, but mutant cells cannot become thermotolerant and die after the same treatment. The mutation is named chp 1 (cell cycle, heat shock, and phosphorylation defect).

Perspectives on tubulin isotype function and evolution based on the observation that Tetrahymena thermophila microtubules contain a single alpha- and beta-tubulin.

We have cloned and sequenced the two beta-tubulin genes of the ciliated protozoan Tetrahymena thermophila. The two genes encode identical 443 amino acid peptides which are 99.7% identical to the beta-tubulin proteins of T. pyriformis and 95% identical to human beta 1 tubulin. T. thermophila contains only one alpha-tubulin gene (Callahan et al., 1984: Cell 36:441-445). Thus, all of the extremely diverse microtubule structures in this unicellular organism can be formed from a single alpha- and a single beta-tubulin peptide. We have also carried out a phylogenetic analysis of 84 complete beta-tubulin peptide sequences. This analysis supports two hypotheses regarding beta-tubulin evolution and function: 1) Multifunctional beta-tubulins are under greater evolutionary constraint than beta-tubulins present in specialized cells or in cells with very few microtubule related functions, which can evolve rapidly; and 2) Cells which form axonemes maintain a homogeneous population of tubulins.