Dwayne Ford

IL‐13 signal transduction in human monocytes: phosphorylation of receptor components, association with Jaks, and phosphorylation/activation of Stats

Interleukin (IL)‐13 regulates monocyte function and is a potent stimulator of 15‐lipoxygenase expression. In different cell types, the functional IL‐13 receptor complex can be comprised of variable protein components and has not been thoroughly examined in human monocytes. Here, we identify the receptor components and upstream signaling events initiated by IL‐13 in primary human blood monocytes. Th expression, phosphorylation and associated Jak kinases of the known, variable receptor components, IL‐4Rα, IL‐2Rγc, IL‐13Rα1 and IL‐13Rα2, were examined. We determined that IL‐4Rα and IL13Rα1 are phosphorylated upon exposure to IL‐13. Although IL‐2Rγc is also expressed, it is not phosphorylated upon exposure to IL‐13. Evaluation of the presence of IL‐13Rα2 failed to reveal significant mRNA or protein expression. Earlier, our laboratory showed that IL‐13 induced the phosphorylation of Jak2 and Tyk2 in monocytes and that expression of both Jaks was essential for downstream signaling by IL‐13. Here, we report that Jak2 is associated with IL‐4Rα, and Tyk2 is associated with the IL‐13Rα1 component of the IL‐13 receptor complex. Additionally, Stat proteins 1α, 3, 5A, 5B, and 6 are phosphorylated in response to IL‐13. Further, the nuclear translocation and DNA binding of each of these Stats were induced by IL‐13. These data represent the first complete report of the functional IL‐13 receptor complex and early signaling events in human monocytes. This information is critical for understanding the IL‐13 response of monocytes in inflammation.

Induction of the IL-13 receptor α2-chain by IL-4 and IL-13 in human keratinocytes : involvement of STAT6, ERK and p38 MAPK pathways

IL-4 and IL-13 are related cytokines which induce both pro- and anti-inflammatory effects depending on the cell type they act upon and the nature of the receptors expressed. The type I receptor complex is composed of the IL-4Rα and γc and only binds IL-4, whereas, in the type II receptor, IL-4Rα dimerizes with IL-13Rα1 upon either IL-4 or IL-13 binding. Another ligand binding chain potentially implicated in the IL-4/IL-13 receptor has been described, the IL-13Rα2, but the regulation of its expression and its role in IL-4/IL-13 transduction is poorly understood. In this study we report that IL-4 and IL-13 upregulate IL-13Rα2 at both the mRNA and protein levels in the keratinocyte cell line HaCaT. In these cells, IL-4 or IL-13 were shown to activate the Janus Kinases JAK1 and JAK2, the transcription factor STAT6, and the ERK and p38 mitogen-activated protein kinases. We show that IL-4 or IL-13-induced IL-13Rα2 mRNA expression was inhibited by the ERK inhibitor U0126, the JAK inhibitor AG490 and, to a lesser extent, the p38 MAPK inhibitor SB203580. Moreover, expression of a constitutive active mutant of STAT6 alone did not modify IL-13Rα2 mRNA expression, but potentiated the effects of IL-4 or IL-13 on IL-13Rα2 expression. The constitutive active mutants of MEK1 or MKK6 increased the level of expression of IL-13Rα2 mRNA even in absence of stimulation. Our findings demonstrate, for the first time, that IL-4 and IL-13 can induce IL-13Rα2 expression in keratinocytes, and that the ERK and p38 MAPK together with JAK2 and STAT6 play a critical role in this process.

Long-term bone marrow culture and its clinical potential in chronic wound healing

Bone marrow‐derived cells have long been regarded to play a crucial role in the homeostasis of skin. We have previously described the clinical benefit of directly applying autologous bone marrow aspirate and cultured bone marrow cells to recalcitrant chronic skin wounds. The initial response to treatment appears to be vascular in nature with the formation of new blood vessels. The difficulty in consistently growing adequate numbers of cells for delivery to patients was, however, a limiting factor. Here, in a subsequent protocol, we describe an improved bone marrow culture system yielding a reliable growth of bone marrow cells and leading to a greater clinical response. Cells expressing markers of endothelial progenitors including CD133, CD146, and particularly CD14 are enhanced in these cultures. CD14‐isolated cells produced colonies in endothelial cell assays and sprouting in matrigel assays. Angiogenic cytokines, including angiogenin, epithelial neutrophil‐activating protein‐78, growth‐regulated oncogene, growth‐regulated oncogene‐α, Interleukin‐8, CXC16, and monocyte chemoattractant protein‐1, were found to be elevated in these cultures. Administration of improved culture cells to patients with chronic wounds present for >1 year lead to an enhanced clinical response.

Cloning and Characterization of Two Promoters for the Human HSAL2 Gene and Their Transcriptional Repression by the Wilms Tumor Suppressor Gene Product

HSAL2 is a member of a gene family that encodes a group of putative developmental transcription factors. The HSAL gene complex was originally identified on the basis of DNA sequence homology to a region-specific homeotic gene (SAL) in Drosophila. This study reveals a novel, functional 5′ exon for HSAL2 and demonstrates that two distinct HSAL2 gene transcripts arise from two overlapping transcription units, resulting in proteins that differ by 25 amino acids. By utilizing functional luciferase reporter assays, two distinct promoters for HSAL2, P1 for the proximal promoter (upstream of exon 1) and P2 for the distal promoter (upstream of exon 1A), were identified. Evaluation of mRNA prevalence and tissue specificity, with particular focus on adult tissues, revealed that production of mRNA from P1 was selective and relatively rare. Production of mRNA from P2 was demonstrably higher and was expressed by a greater number of tissues. In contradistinction,HSAL2 expression directed by P2 was undetectable in some malignant populations as opposed to their normal human counterparts, suggesting a potential role as a tumor suppressor gene. Consensus-binding sites were identified for several transcriptional factors, with multiple sites for WT-1, and Hox-1.3 present within both the P1 and P2 regions. In transient transfection assays, transcription from both HSAL2 P1 and P2 was strikingly repressed by the WT-1 tumor suppressor protein. These findings suggest that an intracellular WT-1/HSAL2pathway may play a role in development and hematopoiesis.

Hsal 1 is related to kidney and gonad development and is expressed in Wilms tumor.

Abstract. Townes-Brocks syndrome (TBS) is a human genetic disorder with features including urogenital, limb, anal and cardiac malformations associated with mutations of the TBS gene, Hsal 1. To begin to understand the role of the Hsal 1 protein (p140) in both normal development and disease pathogenesis, both message and protein expression were evaluated in specific tissues associated with TBS. DNA sequence information for Hsal 1 predicts that this homeotic, Drosophila homologue (Sal) encodes a zinc-finger protein consistent with a transcription factor. mRNA for Hsal 1 was highly expressed in fetal kidney and brain, with detectable production in thymus and heart. p140 was found in fetal ureteric bud, fetal and postnatal renal tubular epithelium, and renal blastema. In the 14-week fetal testis, the Hsal 1 protein was specifically expressed in the testosterone producing Leydig cells while in adult gonads Hsal 1 was also found in both Leydig and Sertoli cells, spermatogonia of the testis, and granulosa cells of the ovary. Evaluation of Wilms tumor revealed consistently high expression of the gene product in the epithelial and blastemal components. These spatial and temporal patterns of expression for Hsal 1, and the phenotypic effects associated with TBS, suggest that Hsal 1 plays an important role in the development and functional maintenance of the kidney and gonads. Furthermore, the Hsal 1 gene product may play a part in the pathogenesis of specific neoplasms occurring in these organs in addition to its specific role in Townes-Brocks syndrome.

Carnitine and Derivatives in Experimental Infections

Energy in humans and higher animals is generated through s-oxidation of long-chain fatty acids, which are transported across the mitochondrial membrane by carnitine (3-hydroxy-4-methyl-ammoniobutanoate). The bulk of body carnitine is found in cardiac and skeletal tissue, and to a lesser extent in various organs such as the liver.1–3 Thus, generating adequate levels of carnitine is an important factor in maintaining normal metabolic processes. 4,5 Indeed, it has been shown that L-carnitine O-palmitoyltransferase deficiency is accompanied by hypoketoic hypoglycemia and cardiomyopathy,6 an observation which may indicate a role for carnitine in cardiac disease.7,9 Metabolic disorders of fatty acids have also been associated with carnitine deficiency. Syndromes and symptoms include liver dysfuction, disorders of the central nervous system, and skeletal muscle weakness.10 In addition to its classical role in energy metabolism, recent studies have provided evidence for an immunomodulatory role for carnitine, particularly in alleviating pathogenic symptoms induced by infectious agents, as discussed below.