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Treatment of AZ-521 cells with Helicobacter pylori VacA increased cyclooxygenase 2 (COX-2) mRNA in a time- and dose-dependent manner. A p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, blocked elevation of COX-2 mRNA levels, whereas PD98059, which blocks the Erk1/2 cascade, partially suppressed the increase. Consistent with involvement of p38 MAPK, VacA-induced accumulation of COX-2 mRNA was reduced in AZ-521 cells overexpressing a dominant-negative p38 MAPK (DN-p38). Phosphatidylinositol-specific phospholipase C, which inhibits VacA-induced p38 MAPK activation, blocked VacA-induced COX-2 expression. In parallel with COX-2 expression, VacA increased prostaglandin E2(PGE2) production, which was inhibited by SB203580 and NS-398, a COX-2 inhibitor. VacA-induced PGE2production was markedly attenuated in AZ-521 cells stably expressing DN-p38. VacA increased transcription of a COX-2 promoter reporter gene and activated a COX-2 promoter containing mutated NF-B or NF-interleukin-6 sites but not a mutated cis-acting replication element (CRE) site, suggesting direct involvement of the activating transcription factor 2 (ATF-2)/CREB-binding region in VacA-induced COX-2 promoter activation. The reduction of ATF-2 expression in AZ-521 cells transformed with ATF-2-small interfering RNA duplexes resulted in suppression of COX-2 expression. Thus, VacA enhances PGE2production by AZ-521 cells through induction of COX-2 expression via the p38 MAPK/ATF-2 cascade, leading to activation of the CRE site in the COX-2 promoter.

ABSTRACTTreatment of AZ-521 cells with Helicobacter pyloriVacA increased cyclooxygenase 2 (COX-2) mRNA in a time- and dose-dependent manner. A p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, blocked elevation of COX-2 mRNA levels, whereas PD98059, which blocks the Erk1/2 cascade, partially suppressed the increase. Consistent with involvement of p38 MAPK, VacA-induced accumulation of COX-2 mRNA was reduced in AZ-521 cells overexpressing a dominant-negative p38 MAPK (DN-p38). Phosphatidylinositol-specific phospholipase C, which inhibits VacA-induced p38 MAPK activation, blocked VacA-induced COX-2 expression. In parallel with COX-2 expression, VacA increased prostaglandin E2(PGE2) production, which was inhibited by SB203580 and NS-398, a COX-2 inhibitor. VacA-induced PGE2production was markedly attenuated in AZ-521 cells stably expressing DN-p38. VacA increased transcription of a COX-2 promoter reporter gene and activated a COX-2 promoter containing mutated NF-κB or NF-interleukin-6 sites but not a mutated cis-acting replication element (CRE) site, suggesting direct involvement of the activating transcription factor 2 (ATF-2)/CREB-binding region in VacA-induced COX-2 promoter activation. The reduction of ATF-2 expression in AZ-521 cells transformed with ATF-2-small interfering RNA duplexes resulted in suppression of COX-2 expression. Thus, VacA enhances PGE2production by AZ-521 cells through induction of COX-2 expression via the p38 MAPK/ATF-2 cascade, leading to activation of the CRE site in the COX-2 promoter.

In order to elucidate the relationship between prekeratin and keratin, we performed pulse‐chase experiments using [35S]methionine (35S‐Met) in vitro. Of 6 prekeratin molecules (49, 52, 55, 62, 69 and 71 kDa) that incorporated 35S‐Met, the 55‐kDa prekeratin incorporated the most 35S‐Met. In 3 molecules (52, 55 and 62 kDa) incorporation was decreased at 30 min after being chased; however, incorporation of only two molecules (55 and 62 kDa) of the 6 prekeratins was increased at 60 min. From these results and our previous data, we conclude that the initial stage of processing is as follows: 3 prekeratin molecules (52, 55 and 62 kDa) are first cleaved in the N‐terminal region, then two prekeratin molecules (55 and 69 kDa) are processed to intermediates (52 and 62 kDa) by some proteolytic enzyme(s).

In order to clarify the initial formation of keratin from prekeratin molecules, we undertook a pulse‐chase experiment using [U‐14C]‐glycine labelled prekeratin subunit mixture in vitro.

Keratin and prekeratin were extracted from human plantar epidermis and carried on SDS‐PAGE. Prekeratin in the basal as well as spinous cell layer of the epidermis was composed of 6–7 bands (M.W.: about 71,000‐49,500), while keratin in the surface of stratum corneum was composed of 3–4 bands (M.W.: about 66,500–53,000), indicating molecular modulation of prekeratin during epidermal cell differentiation.

The [ 14C]Gly-labelled keratin polypeptides extracted with 1% SDS and 10 mM DTT were made to undergo changes with an enzyme fraction (ammonium sulfate, 50–75% saturated fraction) prepared from a human epidermis in the presence of 1% Triton X-100. In particular, 69-67 kDa peptides were considerably decreased with the above enzyme fraction in the time course experiments, and the components strongly bound to the cell membrane had little effect on the above reaction. In addition, in the case of the [ 14C]Gly-labelled keratin filament assembly, 69 and 62 kDa peptides were decreased and 55, 52 and 50 kDa peptides were increased with the same enzyme fraction in the time course experiments. From these results, we estimated that the proteolytic enzyme(s) may exist in the human epidermis, and may be processed to keratin intermediates from prekeratin during the initial stage of terminal differentiation in the human epidermis.

To investigate the process of keratin formation, changes in polypeptide composition of epidermal keratin and of prekeratin were examined using serial slices of human skin. In most of the skin surface, prekeratin was composed of 70K, 58K, 56K, 49.5K and occasionally 55K polypeptides, and an additional 63K polypeptide was present in the skin of the palm and sole. The decrease in molecular weight of these prekeratins to that of keratins was observed in the region of the granular and transient cell layers, especially in that of palm and sole. This was partially confirmed by preliminary digestion experiments in vitro. Our present studies suggest that limited proteolysis of higher molecular weight prekeratin(s) is a step in keratin formation of the stratum corneum.