Effects of promyelocytic leukemia zinc finger protein on the proliferation of cultured human corneal endothelial cells.

PURPOSE: To determine whether the promyelocytic leukemia zinc finger (PLZF) protein, a transcriptional repressor and negative regulator during cell cycling, plays a role in the proliferation of cultured human corneal endothelial cells (HCECs). METHODS: The expressions of the mRNA and the protein of PLZF were determined by real-time PCR and western blot analysis, respectively. The changes in the expression of the PLZF gene of cultured HCECs were investigated at different times after cell-cell contacts were disrupted by incubation with EDTA. The cell proliferation rate was assessed with a real-time cell electronic sensing (RT-CES) system after cultured HCECs were infected with either PLZF or LacZ encoding adenovirus vector (Ad-PLZF or Ad-LacZ). The PLZF-regulating genes were analyzed by DNA microarray analysis in cultured HCECs infected with Ad-PLZF. RESULTS: The expression of the mRNA of PLZF was first detected when the cultured HCECs became confluent, and the relative amount of PLZF mRNA continued to increase for up to 5 days as the cell-cell contacts were formed more firmly. On the other hand, the expression of the mRNA of PLZF decreased about 20 fold 3 h after EDTA exposure, and gradually returned to the original level as the cell-cell contacts were reformed at 72 h after the exposure. The assessment using the RT-CES system showed that the proliferation of cultured HCECs was inhibited for up to 72 h when infected by Ad-PLZF. DNA microarray analysis revealed that the transforming growth factor-beta stimulated clone 22 (TSC-22) gene was up-regulated by 2.32 fold when infected by Ad-PLZF. CONCLUSIONS: These findings indicate that the expression of PLZF in HCECs is closely related to the formation of cell-cell contacts, and that PLZF may play a role in suppressing their proliferation.

ADAM binding protein Eve-1 is required for ectodomain shedding of epidermal growth factor receptor ligands.

A disintegrin and metalloproteases (ADAMs) are implicated in the ectodomain shedding of epidermal growth factor receptor (EGFR) ligands in EGFR transactivation. However, the activation mechanisms of ADAMs remain elusive. To analyze the regulatory mechanisms of ADAM activation, we performed yeast two-hybrid screening using the cytoplasmic domain of ADAM12 as bait, and identified a protein that we designated Eve-1. Two cDNAs were cloned and characterized. They encode alternatively spliced isoforms of Eve-1, called Eve-1a and Eve-1b, that have four and five tandem Src homology 3 (SH3) domains in the carboxyl-terminal region, respectively, and seven proline-rich SH3 domain binding motifs in the amino-terminal region. The short forms of Eve-1, Eve-1c and Eve-1d, translated at Met-371 are human counterparts of mouse Sh3d19. Northern blot analysis demonstrated that Eve-1 is abundantly expressed in skeletal muscle and heart. Western blot analysis revealed the dominant production of Eve-1c in human cancer cell lines. Knockdown of Eve-1 by small interfering RNA in HT1080 cells reduced the shedding of proHB-EGF induced by angiotensin II and 12-O-tetradecanoylphorbol-13-acetate, as well as the shedding of pro-transforming growth factor-alpha, promphiregulin, and proepiregulin by 12-O-tetradecanoylphorbol-13-acetate, suggesting that Eve-1 plays a role in positively regulating the activity of ADAMs in the signaling of EGFR-ligand shedding.

CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

CCL28 is a CC chemokine signaling via CCR10 and CCR3 that is selectively expressed in certain mucosal tissues such as exocrine glands, trachea, and colon. Notably, these tissues commonly secrete low-salt fluids. RT-PCR analysis demonstrated that salivary glands expressed CCL28 mRNA at the highest levels among various mouse tissues. Single cells prepared from mouse parotid glands indeed contained a major fraction of CD3(-)B220(low) cells that expressed CCR10 at high levels and CCR3 at low levels and responded to CCL28 in chemotaxis assays. Morphologically, these cells are typical plasma cells. By immunohistochemistry, acinar epithelial cells in human and mouse salivary glands were strongly positive for CCL28. Furthermore, human saliva and milk were found to contain CCL28 at high concentrations. Moreover, the C terminus of human CCL28 has a significant sequence similarity to histatin-5, a histidine-rich candidacidal peptide in human saliva. Subsequently, we demonstrated that human and mouse CCL28 had a potent antimicrobial activity against Candida albicans, Gram-negative bacteria, and Gram-positive bacteria. The C-terminal 28-aa peptide of human CCL28 also displayed a selective candidacidal activity. In contrast, CCL27, which is most similar to CCL28 and shares CCR10, showed no such potent antimicrobial activity. Like most other antimicrobial peptides, CCL28 exerted its antimicrobial activity in low-salt conditions and rapidly induced membrane permeability in target microbes. Collectively, CCL28 may play dual roles in mucosal immunity as a chemoattractant for cells expressing CCR10 and/or CCR3 such as plasma cells and also as a broad-spectrum antimicrobial protein secreted into low-salt body fluids.