The early signaling pathway of live yeast cell derivative in THP-1 monocytes.

Live yeast cell derivative (LYCD) is a medicinal yeast extract that has been used in the treatment of burns, wounds and hemorrhoids for over 70 years. It has been shown to enhance the closure of skin wounds in diabetic mice by increasing inflammation, angiogenesis, formation of granulation tissue and epithelial migration. An active fraction of LYCD has been identified as a mixture of peptides ranging in size from 5 kDA to 17 kDA. Despite its widespread use over many years, understanding of the mechanism by which LYCD acts to effect tissue repair responses is very limited. In this study, we have used a human monocyte-derived cell line, THP-1, as a representative of the inflammatory component of the wound healing process. We have identified two of the earliest responses to LYCD as an increase in cytoplasmic free calcium ([Ca2+]i) and the transcripts for c-fos. We have found that the increase in [Ca2+]i is both necessary and sufficient to account for the LYCD-induced elevation of c-fos. Furthermore, we have shown that the signaling pathway by which LYCD elevates [Ca2+]i involves both mobilization of Ca2+ from intracellular stores and influx of Ca2+ from the extracellular medium. Mobilization of store Ca2+ occurs first via activation of phospholipase C and this is followed by influx through activation of store operated calcium channels. These results constitute the first delineation of the early steps of the LYCD signaling pathway.

Identification of copper-responsive genes in an early life stage of the fathead minnow Pimephales promelas.

While physiological changes associated with copper toxicity have been studied in adult fathead minnow, Pimephales promelas, little is known about the effect of copper on newly hatched larvae. As a result we initiated an investigation on the mechanism of copper toxicity in 24 h post-hatch larvae using gene expression changes to identify responsive genes. Fish were exposed to copper concentrations of 0, 50, 125 and 200 mug/L in a 48 h toxicity test. Total RNA from survivors was used in a differential display assay to screen for differentially expressed gene products. Altogether, 654 copper-responsive differentially expressed bands were collected. Database searches found homology for 261 sequences. One hundred and sixty-one bands were homologous to NCBI genes of known function, of which 69 were individual genes. The most abundant categories of functional genes responding to copper were involved in protein synthesis/translational machinery and contractile proteins. Twenty-one dose-responsive genes were measured for expression changes using real-time quantitative PCR. Differential gene expression was validated for 11 of 13 genes, when a 1.2 times qPCR difference between the copper and control samples was observed. Transcripts identified as titin, cytochrome b, fast muscle specific heavy myosin chain 4, fast muscle troponin I, proteasome 26S subunit and troponin T3a were induced over twofold. Differential display bands identified as 60S ribosomal proteins L27 and L12 were repressed approximately threefold. We conclude that copper exposure affects several cellular pathways in larval fathead minnows with protein synthesis, ribosome structure, and muscle contractile proteins being the most sensitive to this stress.

Live yeast cell derivative induces c-fos expression in THP-1 monocytes.

Live Yeast Cell Derivative is a medicinal extract of Saccharomyces cerevisiae that has demonstrated efficacy in improving the rate and quality of wound healing in mouse and human systems. However, the mechanisms by which LYCD promotes healing are largely uncharacterized. In this report, we demonstrate that LYCD has effects on the transcriptional profile of the human monocytic cell line THP-1. Thirty minute exposures of THP-1 cells with LYCD induced a 6 to 44-fold, dose-dependent increase in the relative expression of the proto-oncogene c-fos in complete media containing 10% FBS or in low serum media containing 0.1% FBS. Furthermore, protein levels of c-Fos rise at 30 minutes of LYCD exposure and remained detectable for at least 120 minutes of LYCD exposure. However, the relative abundance of the c-fos transcript returned to basal levels by 120 minutes. LYCD also induced expression of c-jun with maximal expression of 3-fold at 60 minutes of exposure. Pretreatments with EGFR kinase inhibitor AG-1478 and the MEK1 inhibitor PD98059 blocked the LYCD-dependent increases in c-fos expression. Consistent with signaling through the EGFR, we have demonstrated by RT-PCR the presence of the mRNA for the EGFR (ErbB1/HER1) in THP-1 cells. Taken together these data suggest that LYCD acts through an EGFR-like cell surface receptor resulting in the activation of the EGFR kinase and the ERK1/2 signaling cascade.