HETEs enhance IL-1-mediated COX-2 expression via augmentation of message stability in human colonic myofibroblasts

Proinflammatory cytokines and eicosanoids are central players in intestinal inflammation. IL-1, a key cytokine associated with intestinal mucosal inflammation, induces COX-2 expression in human colonic myofibroblasts (CMF) and increased prostaglandin E2secretion is associated with inflammatory bowel disease (IBD) and colorectal cancer (CRC). We have previously demonstrated that IL-1α-induced cyclooxygenase-2 (COX-2) expression is the result of NF-κB- and ERK-mediated transcription, as well as COX-2 message stabilization, which depends on p38, MAPKAPK-2 (MK-2) and human antigen R (HuR) RNA binding protein activation. Lipoxygenase (LOX)-derived hydroxyeicosatetraenoic acids (HETEs) are elevated in IBD and colonic adenomas and “cross talk” has been observed between the COX and LOX pathways. Since COX-2 expression is primarily in CMFs in colonic adenomas, we examined the impact of LOX metabolites, particularly HETEs, on IL-1α-induced COX-2 expression in human CMFs. Although 5(S)-, 12(R)-, and 15(S)-HETEs alone had little to no effect on COX-2 expression, they enhanced IL-1-mediated COX-2 expression 3.6 ± 0.5-fold. Studies utilizing heterogeneous nuclear RNA amplification and 5,6-dichloro-β-d-ribofuranosylbenzimidazole treatment were undertaken to measure COX-2 transcription and message stabilization, respectively. We found that HETEs enhanced IL-1-induced COX-2 mRNA levels in CMF as the result of increased p38, MK-2, and HuR activity, increasing message stability greater than that observed with IL-1 alone. Thus HETEs can act synergistically with IL-1α to induce COX-2 expression in human CMFs. HETEs may play a role in both colonic inflammation and in increasing the risk of CRC in IBD independently and via induction of COX-2-mediated prostaglandin secretion.

Activation of the transforming potential of the human fos proto-oncogene requires message stabilization and results in increased amounts of partially modified fos protein

The requirements for activation of the transformation potential of the human c-fos proto-oncogene were investigated. Recombinant plasmids containing the Moloney murine leukemia virus long terminal repeat directing transcription of the c-fos coding region and either the authentic c-fos 3' untranslated region (UTR) or the 3' UTR from human c-myc were inefficient at inducing transformation. In contrast, a recombinant that substituted most of the c-fos 3' UTR with the 3' portion of the simian virus 40 T-antigen gene transformed cells well. This difference in transformation efficiency appeared to be due to significantly higher levels of fos mRNA and protein expressed from the transforming recombinant. This, in turn, was due to the much greater stability of its mRNA compared with those from the poorly transforming recombinants containing the c-fos or c-myc 3' UTR. Thus, the 3' UTR of the human c-fos mRNA is responsible for its rapid degradation and limits the steady-state levels of transcript and protein. Cells transformed by the activated human c-fos plasmids contained increased amounts of partially modified c-fos protein (c-Fos). This form of c-Fos turned over much more rapidly than the highly modified form of c-Fos induced by serum stimulation.

Activation of the transforming potential of the human fos proto-oncogene requires message stabilization and results in increased amounts of partially modified fos protein.

The requirements for activation of the transformation potential of the human c-fos proto-oncogene were investigated. Recombinant plasmids containing the Moloney murine leukemia virus long terminal repeat directing transcription of the c-fos coding region and either the authentic c-fos 3' untranslated region (UTR) or the 3' UTR from human c-myc were inefficient at inducing transformation. In contrast, a recombinant that substituted most of the c-fos 3' UTR with the 3' portion of the simian virus 40 T-antigen gene transformed cells well. This difference in transformation efficiency appeared to be due to significantly higher levels of fos mRNA and protein expressed from the transforming recombinant. This, in turn, was due to the much greater stability of its mRNA compared with those from the poorly transforming recombinants containing the c-fos or c-myc 3' UTR. Thus, the 3' UTR of the human c-fos mRNA is responsible for its rapid degradation and limits the steady-state levels of transcript and protein. Cells transformed by the activated human c-fos plasmids contained increased amounts of partially modified c-fos protein (c-Fos). This form of c-Fos turned over much more rapidly than the highly modified form of c-Fos induced by serum stimulation.

Early retinoic acid-induced F9 teratocarcinoma stem cell gene ERA-1: alternate splicing creates transcripts for a homeobox-containing protein and one lacking the homeobox.

Retinoic acid (RA), the natural acidic derivative of vitamin A, can modulate the expression of specific genes and can induce some cell types, such as the murine F9 teratocarcinoma stem cell line, to differentiate in culture. As an initial step toward understanding the molecular mechanism(s) by which RA exerts these effects, we previously isolated cDNA clones for a gene, ERA-1, which has the characteristics of an early, direct target for RA. We demonstrated that RA causes a rapid, dose-dependent, and protein synthesis-independent expression of the ERA-1 gene (G. J. LaRosa and L. J. Gudas, Proc. Natl. Acad. Sci. USA 85:329-333, 1988). We now report the full-length cDNA sequence and the further characterization of this gene. The data indicate that the RA-induced 2.2- to 2.4-kilobase ERA-1 RNA species that we previously detected consists of two alternately spliced messages. One mRNA encodes a protein with a predicted mass of about 36 kilodaltons (kDa) that possesses the Hox 1.6 homeobox domain. The other mRNA encodes a truncated protein of about 15 kDa which is identical to the 36-kDa protein for 114 amino acids at the amino-terminal end but which lacks the homeobox amino acid sequence. The RA-associated increase in the ERA-1 mRNA level does not appear to be due to message stabilization, suggesting that the response is at the level of transcription. By Northern (RNA) blot analysis, the usual 2.2- to 2.4-kilobase mRNA species was also rapidly expressed in P19 teratocarcinoma cells during their differentiation to fibroblastic cells in response to RA and was detected in day 10.5 and day 13.5 mouse embryos. This result indicates that the expression of this gene is not limited to the endodermal differentiation of F9 cells.

Early retinoic acid-induced F9 teratocarcinoma stem cell gene ERA-1: alternate splicing creates transcripts for a homeobox-containing protein and one lacking the homeobox

Retinoic acid (RA), the natural acidic derivative of vitamin A, can modulate the expression of specific genes and can induce some cell types, such as the murine F9 teratocarcinoma stem cell line, to differentiate in culture. As an initial step toward understanding the molecular mechanism(s) by which RA exerts these effects, we previously isolated cDNA clones for a gene, ERA-1, which has the characteristics of an early, direct target for RA. We demonstrated that RA causes a rapid, dose-dependent, and protein synthesis-independent expression of the ERA-1 gene (G. J. LaRosa and L. J. Gudas, Proc. Natl. Acad. Sci. USA 85:329-333, 1988). We now report the full-length cDNA sequence and the further characterization of this gene. The data indicate that the RA-induced 2.2- to 2.4-kilobase ERA-1 RNA species that we previously detected consists of two alternately spliced messages. One mRNA encodes a protein with a predicted mass of about 36 kilodaltons (kDa) that possesses the Hox 1.6 homeobox domain. The other mRNA encodes a truncated protein of about 15 kDa which is identical to the 36-kDa protein for 114 amino acids at the amino-terminal end but which lacks the homeobox amino acid sequence. The RA-associated increase in the ERA-1 mRNA level does not appear to be due to message stabilization, suggesting that the response is at the level of transcription. By Northern (RNA) blot analysis, the usual 2.2- to 2.4-kilobase mRNA species was also rapidly expressed in P19 teratocarcinoma cells during their differentiation to fibroblastic cells in response to RA and was detected in day 10.5 and day 13.5 mouse embryos. This result indicates that the expression of this gene is not limited to the endodermal differentiation of F9 cells.