Under unwounded conditions, only EGF was present at concentrations near the ligand's Kd for the receptor, indicating it is the primary mediator of corneal epithelial homeostasis. Other ligands were present but at concentrations 11- to 7500-fold less their Kd, preventing significant ligand binding. Further, the high levels of EGF and its predicted binding preclude receptor occupancy by exogenous ligand and can explain the discrepancy between the in vitro and in vivo data. Therefore, therapeutic use of EGFR ligands may be unpredictable and impractical.

Toxoplasma gondii is a ubiquitous apicomplexan parasite that can cause severe disease in fetuses and immune-compromised patients. Rhoptries, micronemes, and dense granules, which are secretory organelles unique to Toxoplasma and other apicomplexan parasites, play critical roles in parasite growth and virulence. To understand how these organelles modulate infected host cells, we sought to identify host cell transcription factors triggered by their release. Early growth response 2 (EGR2) is a host cell transcription factor that is rapidly upregulated and activated in Toxoplasma-infected cells but not in cells infected with the closely related apicomplexan parasite Neospora caninum. EGR2 upregulation occurred only when live parasites were in direct contact with the host cell and not from exposure to cell extracts that contain dense granule or micronemal proteins. When microneme-mediated attachment was blocked by pretreating parasites with a calcium chelator, EGR2 expression was significantly reduced. In contrast, when host cells were infected with parasites in the presence of cytochalasin D, which allows rhoptry secretion but prevents parasite invasion, EGR2 was activated. Finally, we demonstrate that Toxoplasma activation of host p38 mitogen-activated protein kinase is necessary but not sufficient for EGR2 activation. Collectively, these data indicate that EGR2 is specifically upregulated by a parasite-derived secreted factor that is most likely a resident rhoptry protein.

Angiopoietins play a significant role in vascular development and angiogenesis. Both angiopoietin-1 (Ang1) and angiopoietin-2 (Ang2) bind the receptor tyrosine kinase Tie2. However, while Ang1 signaling results in the stabilization of vessel structure, Ang2 has been linked to vascular instability. The ratio of these two Tie2 ligands is thus critical for vascular stability and remodeling. This study identifies a mechanism of growth factor-mediated reduction in Ang2 expression in vascular smooth muscle cells (VSMCs). In response to PDGF, VSMCs downregulated Ang2 mRNA levels by 75% within 4 h, with a subsequent decrease in Ang2 protein levels. Quantitation of endogenous transcription rates revealed that PDGF stimulation did not alter Ang2 transcription rates, but instead induced a posttranscriptional mechanism of rapid Ang2 mRNA destabilization. The Ang2 mRNA half-life was reduced by at least 50% after PDGF treatment. The PDGF-induced mRNA turnover mechanism was dependent on several MAPK pathways, including ERK and JNK. In contrast, IGF-I, which did not significantly activate ERK or JNK, stimulated increased Ang2 expression through transcriptional activation. These findings demonstrate that VSMCs adjust Ang2 expression through multiple mechanisms, including changes in transcription as well as posttranscriptional mRNA destabilization.

No abstract

Toxoplasma gondii is a wide spread pathogen that can cause severe and even fatal disease in fetuses and immune-compromised hosts. As an obligate intracellular parasite, Toxoplasma must alter the environment of its host cell in order to establish its replicative niche. This is accomplished, in part, by secretion of factors into the host cell that act to modulate processes such as transcription. Previous studies demonstrated that genes encoding transcription factors such as c-jun, junB, EGR1, and EGR2 were amongst the host genes that were the most rapidly upregulated following infection. In cells stimulated with growth factors, these genes are regulated by a transcription factor named Serum Response Factor. Serum Response Factor is a ubiquitously expressed DNA binding protein that regulates growth and actin cytoskeleton genes via MAP kinase or actin cytoskeletal signaling, respectively. Here, we report that Toxoplasma infection leads to the rapid activation of Serum Response Factor. Serum Response Factor activation is a Toxoplasma-specific event since the transcription factor is not activated by the closely related protozoan parasite, Neospora caninum. We further demonstrate that Serum Response Factor activation requires a parasite-derived secreted factor that signals via host MAP kinases but independently of the host actin cytoskeleton. Together, these data define Serum Response Factor as a host cell transcription factor that regulates immediate early gene expression in Toxoplasma-infected cells.

Early gestation mammalian fetuses possess the remarkable ability to heal cutaneous wounds in a scarless fashion. Over the past 20 years, scientists have been working to decipher the mechanisms underlying this phenomenon. Much of the research to date has focused on fetal correlates of adult wound healing that promote fibrosis and granulation tissue formation. It is important to remember, however, that wound repair consists of a balance between tissue synthesis, deposition, and degradation. Relatively little attention has been paid to this latter component of the fetal wound healing process. In this study, we examined the ontogeny of ten matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in nonwounded fetal rat skin and fibroblasts as a function of gestational age. We used a semiquantitative polymerase chain reaction protocol to analyze these important enzymes at time points that represent both the scarless and scar-forming periods of rat gestation. The enzymes evaluated were collagenase-1 (MMP-1), stromelysin-1 (MMP-3), gelatinase A (MMP-2), gelatinase B (MMP-9), membrane-type matrix metalloproteinases (MT-MMPs) 1, 2, and 3, and TIMPs 1, 2, and 3. Results demonstrated marked increases in gene expression for MMP-1, MMP-3 and MMP-9 that correlated with the onset of scar formation in nonwounded fetal skin. Similar results were noted in terms of MMP-9 gene expression in fetal fibroblasts. These results suggest that differences in the expression of these matrix metalloproteinases may have a role in the scarless wound healing phenotype observed early in fetal rat gestation. Furthermore, our data suggest that the differential expression of gelatinase B (MMP-9) may be mediated by the fetal fibroblasts themselves.

No abstract

The epidermal growth factor receptor (EGFR) has been shown to be necessary and sufficient for mediating corneal epithelial homeostasis and wound healing. Experimentally, activation of EGFR by epidermal growth factor (EGF), increases healing in a corneal wound; however, therapeutic use of EGF does not improve corneal healing in patients. This study looks at wound healing elicited by other EGFR ligands, specifically betacellulin (BTC). Using human telomerase‐immortalized corneal epithelial cells (hTCEpi) in an in vitro wound healing assay, BTC was shown to be more efficacious than EGF during wound closure. Since BTC can also stimulate ErbB4, an EGFR family member, the efficacy of BTC may be due to activation of this receptor population. However, mRNA and protein levels were assessed in both hTCEpi and primary human corneal epithelial cells and revealed the absence of ErbB4. Differences in ligand binding properties could also explain BTC efficacy. Using radiolabeled ligands to assess binding sites, ligand affinity, pH sensitivity and competition revealed differences between BTC and EGF in these areas. We conclude that the increased efficacy seen with BTC over EGF is not from stimulation of ErbB4 but is, at least in part, from differences in binding properties. Funding: NIH 1R01GM092874 and P20 RR 017703.