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Cell cycle-dependent expression of cyclooxygenase-2 in human fibroblasts¹

Vascular Biology Research Center and Division of Hematology, Department of Medicine, University of Texas-Houston Medical School, Houston, Texas, USA; and Vascular Biology Program, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan

²Correspondence: Vascular Biology Research Center and Division of Hematology, University of Texas-Houston Medical School, 6431 Fannin, MSB 5.016, Houston, Texas 77030-1503, USA. E-Mail: Kenneth.K.Wu{at}uth.tmc.edu

SPECIFIC AIMS

Cyclooxygenase-2 (COX-2) expression is a critical part of inflammation and plays a major role in defending against exogenous stimuli, while its over-expression causes cells to exhibit tumor phenotypical changes. It is unclear how COX-2 expression is regulated. We postulate in this study that COX-2 expression is regulated in a cell cycle-dependent manner: Cells in the G₀ state are highly responsive to COX-2 induction by exogenous stimuli, whereas COX-2 expression in cells entering the growth phase is under tight control. To test this hypothesis, we determined the level of COX-2 expression and promoter activity induced by phorbol 12-myristate 13-acetate (PMA) and interleukin-1ß (IL-1ß) at G₀ and at various phases of cell cycle in a human fibroblast model.

PRINCIPAL FINDINGS

1. Induction of COX-2 expression in G₀ fibroblasts
Human foreskin fibroblasts (HFF) cultured in medium deprived of fetal bovine serum (FBS) for 24 h were subjected to flow cytometry. Ninety percent of the cells were in G₀/G₁ phase, 3% were in S phase, and 7% were in G₂/M phase. Fibroblasts in G₀ phase are distinguished from those in G₁ by the time (12–16 h vs. 6–8 h) they take to enter the S phase. To determine whether a majority of our 24 h serum-starved HFF were in G₀ phase, we added 10% FBS to the serum-deprived cells and assessed the duration of time for these cells to enter the S phase. The FBS-treated cells started to enter the S-phase at 16 h, thus consistent with being in the G₀ state. IL-1ß (1 ng/ml) did not drive the serum-starved cells into cell cycle, and yet it induced COX-2 mRNA and protein expression in a time-dependent manner. Prostaglandin E₂ production was concordantly increased. Similarly, PMA (100 nM) did not drive the cell cycle but induced COX-2 mRNA and protein expressions in a time-dependent manner. COX-2 mRNA and protein induction by IL-1ß persisted beyond 24 h after addition of IL-1ß, while COX-2 expression induced by PMA completely dissipated after 12 h. These results indicate that COX-2 is highly inducible by PMA and IL-1ß in quiescent HFF. Next, we determined whether other pro-inflammatory mediators were similarly induced in G₀ fibroblasts. We measured basal and stimulated levels of matrix metalloproteinase-1 and transforming growth factor ß1 by enzyme immunoassay and heme oxygenase-1 by Western blot analysis. These proteins were undetectable in unstimulated and PMA and IL-1ß–stimulated cells. Induction of COX-2 in G₀ cells may be mediated by a selective mechanism.

2. COX-2 expression in G₀-arrest cells is more robust than that in cycling cells
Adding 2.5% FBS to serum-starved HFF drove the cells to enter S phase at 20 h; more than 50% of the cells were in S phase at 24 h. The level of COX-2 mRNA induced by 2.5% FBS alone was only slightly above the background at 4 h and undetectable at 24 h). Thus, we chose this as a model to determine cell cycle-dependent COX-2 expression. The kinetics of COX-2 mRNA expression in response to PMA stimulation was determined at G₀ (24-h serum-starved cells) and at several representative points of the cells cycle: early G₁ (4-h post serum), mid-G₁ (12-h post serum), S (24 h post serum), and G₂/M (30-h post serum). At each of these time points, HFF were treated with fresh serum-free medium containing PMA and COX-2 mRNA levels were measured at 2, 4, 8, 12, 16 and 24 h after PMA treatment. The time course of COX-2 mRNA induction by PMA was comparable at each cell cycle time point, and maximal COX-2 induction was noted at 8 h. However, the intensity of COX-2 induction declined significantly at 12 h and declined progressively thereafter. Maximal COX-2 mRNA induction at each point of cell cycle was compared by using GAPDH mRNA as the internal control. COX-2 mRNA at early G₁ (4 h) was not significantly different from that at G₀ phase, but the level at mid-G₁ (12 h), S (24 h), and G₂/M (30 h) was reduced to 76%, 46%, and 30% of that of the G₀ level, respectively (Fig. 1a) .

View larger version (14K): [in this window] [in a new window]   Figure 1. Cell cycle-dependent expression of COX-2. FBS for 4 h and 24 h, at which times cells were washed and incubated in fresh medium in the presence or absence of PMA or IL-1ß for 2 h and COX-2 and GAPDH mRNA was blotted with their respective probes. a) Comparison of COX-2 mRNA to GAPDH mRNA ratio at maximal COX-2 expression at representative points of cell cycle after 2.5% FBS treatment or b) COX-2 promoter activity induced by PMA in G0 (0 h), early G1 (4h), and S (24 h) phases of cell cycle. **denotes P<0.01 when compared with G0 values.

3. Level of COX-2 induction in asynchronized HFF is lower than that in G₀-arrested HFF
Considering that previous reports of COX-2 induction by PMA were performed in cells in the presence of 10%–20% FBS in which the cells are asynchronized in cell cycle, we compared COX-2 mRNA expression in response to PMA stimulation in 24 h serum-starved cells versus cells cultured in the presence of 10% FBS. The results show that the COX-2 mRNA level was reduced markedly in asynchronized cells when compared with G₀ arrested cells. These results confirm that COX-2 expression is maximal in G₀ arrested cells.

4. PMA-induced COX-2 promoter activity in G₀ cells was higher than that in S phase cells
A 0.9 kb 5’-flanking fragment (-891 to +9) of human COX-2 promoter was constructed into a promoter-less luciferase expression vector and expressed in HFF. Cells in G₀, early G₁ (4 h post serum), and S (24 h post serum) were stimulated with PMA and luciferase activity was measured. A basal promoter activity was detected at each time point. After subtracting the basal activity, the PMA-stimulated promoter activity at 4 h was slightly higher than that at 0 h but the difference was statistically insignificant. In contrast, the activity at 24 h was markedly reduced (Fig 1b) .

CONCLUSION

COX-2 expression is induced by a myriad of pro-inflammatory, mitogenic, and oncogenic factors. Its expression affords an important defense against environmental insults in organs and tissues such as the arterial wall, gastrointestinal tract, and kidneys. Paradoxically, its over-expression is a key component in several pathological processes such as inflammation, tissue injury, and tumorigenesis. The expression of COX-2 must be regulated in a coordinated manner, otherwise cells and tissues will have detrimental consequences. Little information in the literature addresses this issue. In this study, we have shown that COX-2 expression is regulated in a cell cycle-dependent manner: It is highly induced by IL-1ß and PMA in quiescent (G₀) fibroblasts, but is progressively suppressed after the cells have entered the committed G₁ phase of the cell cycle. COX-2 mRNA levels are reduced by 53% and 70% when a majority of the cells have entered the S and G₂/M phases of cell cycle, respectively. The levels of COX-2 mRNA expressed at S and G₂/M correlated with the percentage of cells in the G₀/G₁ phase, suggesting that cells in the quiescent phase are the predominant responder to exogenous stimuli in mounting COX-2 expression. This notion should be confirmed by determining COX-2 expression in pure fractions of cells at different phases of cell cycle. A vast majority of cells in the human body—including endothelial cells, fibroblasts, macrophages—are in the quiescent state; they are the front line of defense against environmental insults. Our results indicate that the quiescent cells accomplish this mission through COX-2 expression with consequent synthesis of physiologically relevant prostanoids. When excessive COX-2 expression occurs as a result of overwhelming stimuli, COX-2 becomes a pivotal player in inflammation and tissue injury. It has recently been shown that fibroblasts within or adjacent to a tumor mass over-express COX-2, which plays an important role in angiogenesis and tumor growth. These fibroblasts are likely to be in the quiescent state. Thus, cells in the quiescent state may be involved in diverse pathophysiological processes.

An interesting finding of this report is that, although COX-2 is highly inducible, several other inflammatory mediators are not inducible in G₀ cells. The mechanism for selective COX-2 induction in G₀ cells is unclear. Normal cells exit the cell cycle and enter quiescence upon serum withdrawal, nutrient deprivation, or contact inhibition. Such G₀ cells decrease in size because their protein and RNA molecules are degraded and are not rapidly resynthesized. Macromolecular syntheses and enzyme activities are lower in G₀ cells than in proliferating cells. However, despite this apparent dormant state, G₀ cells have distinct genetic features. For instance, E2F-p130 transcriptional repressor complex, statin, growth arrested-specific genes, and von Hippel-Lindau tumor suppressor gene are all highly expressed in G₀ cells and are believed to play an important role in cell cycle arrest. This finding indicates that, despite having a less active protein synthetic capacity than proliferating cells, G₀ cells express a series of unique genes that control highly specific functions. In this report we have demonstrated that G₀ cells are capable of expressing abundant COX-2 in response to exogenous stimuli. We have observed similar results in serum-starved human endothelial cells and murine macrophages. This observation is very important because numerous cell types in the human body are in the G₀ phase. These cells constantly encounter exogenous stimuli. By being able to mount a robust COX-2 response to the environmental insult, they provide the first line of defense. Selective induction of COX-2 and possibly other functionally related genes in G₀ cells represent a new paradigm of cell physiology.

It has been suggested that COX-2 may cause oxidative stress to DNA. When cells enter the proliferative phase, actively replicating DNA is probably more susceptible to oxidative stress and must be protected from the stress. How the DNA is protected from oxidative damage is largely unknown. Our results suggest that suppression of COX-2 transcription represents an important protective mechanism. Results from a previous report and from our unpublished data indicate that COX-2 transcriptional activation by PMA and IL-1ß requires binding of C/EBPß to COX-2 promoter. Suppression of COX-2 transcription as cells enter the proliferative phase is most likely due to an altered expression of C/EBPß or C/EBPß binding activity by post-translational modification of C/EBPß.

In summary, we have found that COX-2 expression in response to PMA and IL-1ß induction is more robust in cells at the G₀ phase than that in cycling cells. There is a progressive decline in COX-2 expression after the cells have entered the G₁ phase of cell cycle. These results suggest that G₀ cells, which constitute a large cell population in the human body, play an important role in inflammation. Results from our study further suggest that COX-2 expression in cycling cells is tightly controlled to avoid excessive oxidative DNA damage. Derangement of this control mechanism may lead to DNA damage and tumorigenesis (Fig. 2 ).

View larger version (19K): [in this window] [in a new window]   Figure 2. Schematic representation of the hypothesized cell cycle-mediated regulation of COX-2 expression. G0 cells demonstrate a robust COX-2 expression not seen in proliferating cells. This hypothesis suggests that the disparity involves a regulatory system, acting at the level of transcription, which allows COX-2 expression to occur in G0 cells, as part of a defense mechanism, but prevents its expression where tumorigenesis is likely to occur. The box with a question mark denotes the unknown function of COX-2 in proliferating cells.

FOOTNOTES

¹ To read the full text of this rticle, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0573fje To cite this article, use (December 8, 2000) FASEB J. 10.1096/fj.00-0573fje

³ These authors contributed equally.

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This Article Full Text (PDF) All Versions of this Article: 15/2/288 00-0573fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by GILROY, D. W. Articles by WU, K. K. Search for Related Content PubMed PubMed Citation Articles by GILROY, D. W. Articles by WU, K. K.