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Effect of prostaglandins on cyclic nucleotide levels in cultured keratinocytes
PROSTAGLANDINS
EFFECT OF PROSTAGLANDINS ON CYCLIC NUCLEOTIDE LEVELS IN CULTURED KERATINOCYTES
David I. Wilkinson, Ph.D. and Elaine K. Orenberg, Ph.D. Department of Dermatology Stanford University School of Medicine Stanford, California 94305 ABSTRACT Guinea pig ear epidermal cells (keratinocytes) were established in primary cultures using trypsin, and treated in their proliferative phase ofgrowth with prostaglandins El, DI, FI~, E2, D2, or F2~. This phase is induced by the addition of retinoic acid during cell plating. Intracellular content of cAMP and cGMP was measured by radioimmunoassay at various times after treatment. Maximum stimulation of cAMP levels was observed with PGD2, smaller increases with PGE2 and relatively transient rises with PGF2~ which were of low significance, but confirm earlier data. Similar results were observed with PGDI, PGEI, and PGFI~ with smaller increases. The effects of D and E PGs were biphasic. Significant increases in cGMP were immediately observed with PGD 2 and PGE2. With PGF2e, maximum cGMP levels were noted after some delay. All PGs tested showed some effect in elevating cyclic nucleotides in keratinocytes. The most striking result was the increase in cAMP on PGDz treatment. INTRODUCTION A number of studies in recent years have suggested that an elevation of cyclic adenosine -3',5'- monophosphate (cAMP) levels in mammalian epidermis takes place during the maturation (keratinization) process. Failure to achieve the necessary intracellular levels apparently may permit epidermal cells (keratinocytes) to remain in a proliferative compartment. This may be accompanied by a possible concomitant increase in cyclic guanosine -3', 5'-monophosphate (cGMP) levels. It has been suggested that the clinical expression of these conditions is the skin disease psoriasis (i). Factors which elevate epidermal cAMP levels include prostaglandins (PGs) among other compounds (2). As most of the cutaneous PG synthetase system is concentrated in the epidermis (3), it seems possible that epidermal PGs play a significant role in the maintenance of cAMP levels. Epidermis can synthesize PGs of the E,F, and
MARCH 1979 VOL. 17 NO. 3
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PROSTAGLANDINS
D series (3,4). As a general rule, PGE species increase cAMP levels (5) and in vitro studies have shown that this also holds for human (6) and guinea pig (7) epidermis, while PGFI~ and PGFe~ are claimed to be wholly ineffective (6) or marginally effective (7). The regulation of cGMP levels in epidermis is not understood. The effect of PGs on this parameter has not been studied. Reports have indicated that PGF species may raise cGMP levels in, for instance, bovine venous strips (8) or cultured fibroblasts (9). On the other hand, a number of studies have appeared which found that PGEI and E2 induce DNA synthesis in mouse epidermis in vitro (i0) and in human (ii) and guinea pig epidermis in vivo (12). These results are difficult to interpret, because the elevation of cAMP in cultured keratinocytes results predictably in decreased DNA synthesis and mitotic inhibition (13). Further, another report found that PGs of the E and F classes had virtually no effect on 3H-thymidine uptake by cultured human keratinocytes, even at 50 DM concentrations (14). The present study has explored the effects of various PGs on cAMP and cGMP levels of cultured guinea pig keratinocytes. In particular, information regarding the following was desired: i. The effect of PGF2~ on cGMP levels. 2. The effect of PGDI and PGD2 on cAMP levels. Data on the effects of PGD species on the epidermis are not available. METHODS Tissue Treatment: Adult albino guinea pigs, Hartley strain, were obtained from Simonsen Farms, Gilroy, California. The animals were sacrificed by exsanguination and their ears removed and depilated by a 3 min treatment with Neet R (Whitehall Laboratories, Inc,, N.Y.), then thoroughly rinsed with water and scrubbed with Septadyne R (Winthrop Laboratories, N.Y.) at 1:5 v/v dilution. The ears were then rinsed briefly with 70% alcohol and used immediately. Cell Culture: Ears were cut radially into 3 pieces and planed with a Castroviejo keratotome set at 0.2 mm. Tissue so obtained was placed in GKN solution containing trypsin (0.3%) for 18-20 min at 37°C in a humidified CO 2 incubator. The epidermal and dermal moieties were isolated and agitated in the culture medium which was McCoy's 5a (modified) containing fetal calf serum (9%), Hepes (15 mM), retinoic acid (5 ~M), gentamicin (50 ~g/ml) and chlortetracycline (50 ~g/ml), pH 7.15. Cells were suspended in this medium at a density of 1.0 x 106 cells/ml and plated in 3.5 em plastic Petri dishes (Lux Scientific) at a density of 2 x 106 cells per dish. Cells were maintained at 37°C in 5% CO 2 atmosphere in a humidified incubator. After 16 hrs, the media was aspirated and replaced by media without retinoic acid. Treatment with PGs: At 48 hours, when the keratinocytes had virtually filled the dishes, the medium was removed and replaced with medium without serum (2.0 ml/dish). Incubation was continued for 3 hours, when an additional 1 ml of serum-free medium was added con-
420
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PROSTAGLANDINS
taining one of the following: PGFI, PGE2, PGFI~, PGFz~,PGDI, PGD2 to give final concentrations of 5 ~M. PGs were dissolved in ethanol before use. Final ethanol concentration was 0.1% v/v. Control dishes received ethanol alone. The dishes were maintained in the incubator. Cyclic nucleotide determinations: At various times after PG addition (time = zero) up to 150 min, dishes were harvested in triplicate by aspirating medium, rinsing the cells with 0.9% saline at 37°C, and addition of ice-cold TCA (6%). The saline rinse and TCA addition required 5 secs. Dishes were scraped off and cells homogenized in TCA using a small teflon/glass homogenizer. The suspensions were centrifuged (i0,000 g, 20 min), the supernatant extracted 4 times with twice its volume of water-saturated ether, and then lyophilized. The residue was dissolved in buffer, and appropriate dilutions acetylated and subjected to radioimmunoassay by the method of Harper and Brooker (15). Materials for radioimmunoassay were obtained from Collaborative Research. Cyclic AMP antiserum was a gift from Dr. Gary Brooker. Prostaglandins were obtain from Dr. J. Pike, Upjohn Co. The pellet from the centrifugation was dissolved in 0.5N NaOH and DNA estimated by the method of Kissane and Robins (16). RESULTS Radioimmunoassay yalidation: Duplicate aliquots were taken from each sample for assay, and the cyclic nucleotide content of each did not usually differ by more than 5% of the mean. Internal standards of cAMP and cGMP (i0, 25 a n d 5 0 femtomoles) were added to a number of samples in each experiment. In a typical series (n=15), 100.9 ± 3.47 (SEM)% of added cAMP and 98.7 ± 1.03% of cGMP was accounted for in subsequent assays. Further validation of the assays was obtained by analysis of randomly selected samples at 2 different dilutions, and by analysis before and after treatment of samples with cyclic nucleotide phosphodiesterase (beef heart, Sigma). With the latter, all material binding to antiserum was destroyed. Recovery of both nucleotides was estimated by carrying aliquots of tritiated cAMP and cGMP through the extractive processes from cell disruption to lyophilization. This was found to be 82.0 ± 1.36% for cAMP and 82.5 ± 1.02% for cGMP (means± SEM; range 73.4-89.4%; n =27). As cGMP was assayed in the presence of cAMP, the binding of cAMP by cGMP antiserum was carefully examined, for each batch of antiserum. It was found that the latter did not react detectably with cAMP in tubes containing less than 1200 femtomoles of cAMP. With the exception of samples obtained using PGD 2 at 5-20 min, none of our samples contained more than 500 fmoles/tube (i00 fmoles in diluted samples). Furthermore, there was good agreement between diluted and undiluted samples. It was concluded that increases in cGMP could not be ascribed to excess cAMP.
MARCH 1979 VOL. 17 NO. 3
421
PROSTAGLANDIN S
Cyclic AMP: iControl values showed an immediate inital decline to somewhat lower values which commenced to rise again after about 60 min to maximum values at 90-120 min, before falling again (Fig. #i). Treatment of cells with PGEI or E2 elicited a rise to significantly high values at 5 or i0 min. M a x i m u m response was observed with PGD I or PGD 2 which provoked threefold and sevenfold increases respectively over the controls. The response to E and D PGs was biphasic. After a decrease, cAMP levels again rose to high levels at 45-75 min, before falling again to or near control values. PGFI~ and PGF2~ caused a slower, smaller rise in cAMP levels, which were not particularly significant for PGFI~ (for instance, p < 0.02 at Figure #I: Variation of cAMP ( - - O - - - - ) and cGMP 6-.-~ ..... ) levels of cultured keratinocytes with time after treatment with PGs as indicated. Control dishes of cells (cAMP, • • ; cGMP ---- • . . . . S - - - - ) did not receive PGs. Each point is the mean ± SEM of 4-6 determinations from 2 experiments. Asterisks indicate the statical significance (student "t" test) over the control value for the point in question: *** ** *
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Table#1: S t a t i s t i c a l evaluation of significance over control values of selected points from Fig. #I for cAMP (top half of Table) and cGMP (bottom half of Table) at the times (min) indicated. Figures are p values from one-sided student " t " test, and were confirmed using Wilc0xon rank test.
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MARCH 1979 VOL. 17 NO. 3
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425
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30 min). These increases due to PGF were relatively transient, returning to control values after 60 min without showing biphasic behavior. In general, PGE2, D2 and F2~ caused greater responses than those observed with PGEI, DI and FI~. Cyclic GMP: As in the case of cAMP, control values showed a fall followed by an increase to higher levels and a final decrease (Fig. #i). A rather more complex reaction to treatment with PGs was observed. PGEI and Ee produced an immediate rise to about twice control levels which fell again in about 60 min. These rises showed good statistical significance (e.g., with PGE2, p < 0.001 at i0 min). PG~I and D2 showed rises of similar magnitude. For PGDI the increase was significant at i0 min, (p < 0.01) but not at any other time point. PGD2 provoked a significant rise at i0 min, (p < 0.001) and levels in PGD2-treated cultures remained generally higher than those of the control dishes until 135 min after the start, but with only indifferent significance. PGFI~ and F2~ produced immediate responses which quickly faded but showed later rises (at 45-60 and at 120 min). The increase seen with PGFe~ showed maximum significance (p < 0.001) at 60 min. DISCUSSION The most striking finding in this study was that all six PGs tested provoked a positive response in terms of both cyclic nucleotides. These responses differed widely in degree. For cAMP, the results agree well with earlier data provided by Adachi who found that'at 5 min after treatment, PGE2 at 3 ~M increased cAMP levels in pig epidermal slices by a factor of 3.2, while PGEI was less effective (2.5-fold increase) and PGF2~ gave a 1.3-fold increase which was judged to be non-significant (17). Interestingly, Adachi also studied PGAI and PGA2 and obtained small, non-significant increases in cAMP. A more surprising result is our finding of a short-term elevation in cAMP due to PGFI~ and PGF2~. Usually these agents are regarded as ineffective, although an earlier paper from this laboratory showed that both increased cAMP accumulation in guinea pig epidermis but with poor statistical significance, p < 0.01 (7). Thus three studies have shown that PGF2~ raises epidermal c~.~ by non-significant (17), marginal (7) or significant amounts (this study)~ The overall impression is that all PGs exert positive effects on epidermal cAMP. Larger increases can be observed at higher concentration; for instance, Adachi reported an 8-fold increase with PGE 2 at 25 ~M (6). Another important finding was that keratinocytes are more sensitive to PGD2 than any other PG. This suggests that if maturation is accompanied by rising cAMP levels, a tenet which is probably true, PGD 2 may be a significant agent in the process of keratinization. Experiments are in progress to determine the effects of PGD 2 on keratinocyte growth or differentiation in culture. For reasons discussed elsewhere (3) the presence of PGD 2 and its biological effects have gone unnoticed in studies of PG-keratinocyte interactions to date.
426
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All PGs tested provoked some increase in cGMP levels above control levels. In the first minutes after PG addition, cAMP and cGMP levels rose together and in the same direction. This is exactly the opposite behaviour to that usually encountered when studying the fluctuations of the cyclic nucleotides in a wide variety of situations such as the progress of synchronized cells (fibroblasts) through the cell cycle (18) or the passage of fibroblasts in or out of quiescence (19), when cAMP and cGMP vary in inverse proportion. However, this unindirectional reaction to PGs has been observed in some cases; Peters has shown that PGEI causes a simultaneous rise in both cAMP and cGMP in embryonic mouse fibroblasts, peaking at i0 and 5 minutes respectively (20). Since increase in cGMP is usually associated with cellular proliferative phenomena, it is tempting to explain recent reports of in vivo epidermal DNA synthesis and mitotic behaviour as due to an increase in cGMP following injection of high local levels of PGEz (10-12). However, W e have observed a burst of 3H-thymidine uptake into DNA in cultured keratinocytes after treatment with PGE 2 but without any measurable increase in amount of DNA per dish (unpublished observation). We have not been able to demonstrate growth in cultures treated with PGs. It is noteworthy that even the control levels of cAMP and cGMP fluctuate in the same direction and in a similar manner during the 2.5 hours of these experiments. This coordinated fluctuation of cAMP and cGMP in keratinocyte cultures (not subjected to treatment with drugs or other agent) is a phenomenon which we have frequently encountered and is the subject of a continuing study. Presumably the variations are related to growth patterns of the cells at 48 hours in their life in culture. The plating efficiency in primary cultures of keratinocytes is maximized by retinoic acid which also applies a mitogenic stimulus (21). The present experiments were carried out at 48 hours after plating because confluence is almost achieved at this point, but the confluent monolayer is followed by multilayer formation, stratification and the onset of keratinization. Thus, under our conditions, at 48 hours the cell population is predominantly proliferative. Serum starvation for 3 hours did not affect the cells; the same response to PGD2 has been observed after preincubation periods of from 0 to 4 hours. It is possible that a more mature (differentiating) population may react otherwise, and species differences may occur. For instance, Harper has found that human keratinocytes growing from explant skin are apparently insensitive to PGs although PGD 1 or PGD 2 was not studied (14). The reasons for the biphasic response to PGE and PGD species are unknown. It seems unlikely that the effect may be due to heterogeneity in the keratinocyte population, because a biphasic response has been reported elsewhere with cultured fibroblasts (a homogenous population) after treatment with PGEI at 5~M (22). The stability of the added PGs was studied by adding tracer amounts
MARCH 1979 VOL. 17 NO. 3
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PROSTAGLANDINS
of aH-PGEz and 3H-PGFz~ to test dishes of cells and observing recovery after 2.5 hours, by extraction of the acidified contents and TLC as before (3). There was no detectable degradation of the tracers. ACKNOWLEDGEMENT This study was supported by N.I.H. grant number AM 15107. REFERENCES i. Voorhees, J.J., N.H. Colburn, M. Stawiski, E.A. Duell, M. Haddox, N.D. Goldberg. Imbalanced cyclic AMP and cyclic GMP levels in the rapidly dividing, incompletely differentiated epidermis of psoriasis. In: Control of Proliferation in Animal Cells (C. Clarkson and R. Baserga, eds). Cold Spring Harbor Laboratory, New York, 1974, p. 635. 2. Iizuka, H., K. Adachi, K.M. Halprin, V. Levine. Adenosine and adenine nucleotides stimulation of skin (epidermal) adenylate cyclase. Biochim. Biophys. Acta 444:695. 1976. 3. Wilkinson, D.I., J.T. Walsh. Prostaglandin biosynthesis in the epidermis and dermis of young mouse skin, and the effects of calcium and cyclic nucleotides. J. Invest. Dermatol. 68:210. 1977. 4. Kingston, W.P., M.W. Greaves. Factors affecting prostaglandin synthesis by rat skin microsomes. Prostaglandins 12:51. 1976. 5. Samuelsson, B., E. Granstrom, K. Green, M. Hamburg, S. Hammarstrom. Prostaglandins. Ann. Rev. Biochem. 44:669. 1975. 6. Adachi, K., K. Yoshikawa, K.M. Halprin, V. Levine. Prostaglandins and cyclic AMP in epidermis. Brit. J. Dermatol. 92:381. 1975. 7. Aso, K., E.K. Orenberg, E.M. Farber. Reduced epidermal cyclic AMP accumulation following prostaglandin stimulation: its possible role in the pathophysiology of psoriasis. J. Invest. Dermatol. 65:375. 1975. 8. Dunham, E.W., M.K. Haddox, N.D. Goldberg. Alteration of vein 3',5' - nucleotide concentrations during changes in contractility. Proc. Nat. Acad. Sei. U.S.A. ~:815, 1974. 9. deAsua, J.L., D. Clingan, P.S. Rudland. Initiation of cell proliferation in cultured mouse fibroblasts by PGF2~. Proc. Nat. Acad. Sci. U.S.A. 72:2724. 1975. i0. Bem, J.L., M.W. Greaves. Prostaglandin El effects on epidermal cell growth in vitro. Arch. Dermatol. Forsch. 251:35. 1974. ii. Eaglstein, W.H., G.D. Weinstein. Prostaglandin and DNA synthesis in human skin. Possible relationship to ultraviolet light effects. J. Invest. Dermatol. 64:386. 1975. 12. Bentley-Phillips, C.B., H. Paulli-Jorgensen, R. Marks. The effects of prostaglandins El and F2~ on epidermal growth. Arch. Dermatol. Res. 257:233. 1977. 13. Flaxman, B.A., R.A. Harper. In vitro analysis of the control of keratinocyte proliferation in human epidermis by physiological and pharmacologic agents, j. Invest. Dermatol. 65:52. 1975.
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14.
15.
16.
17.
18.
19. 20.
21. 22.
Harper, R.A. Effect of prostaglandins on 3H-thymidine uptake into human epidermal cells in vitro. Prostaglandins 12:1019. 1976. Harper, J.F., G. Brooker. Femtomole sensitive radioimmunoassay for cAMP and cGMP after 2'0 acetylation by acetic anhydride in aqueous solution. J. Cyclic Nucleotide Res. 1:207. 1975. Kissane, J., E. Robins. The fluorometric measurement of deoxynucleic acid in animal tissues with special reference to the central nervous system. J. Biol. Chem. 233:184. 1958. Adachi, K. Epidermal cyclic AMP system and its possible role in proliferation and differentiation. In: Biochemistry of Cutaneous Epidermal Differentiation (eds. M. Seiji, I.A. Bernstein ) Univ. Tokyo Press, 1977, p. 336. Seifert, W.E., T.S. Rudland. Cyclic nucleotides and cell growth in cultured mouse cells: correlation of changes in intracellular 3',5'-cGMP concentration with a specific phase of the cell cycle. Proc. Nat. Acad. Sci. U.S.A. 71:4920. 1974. Friedman, D.L. Role of cyclic nucleotides in cell growth and differentiation. Physiol. Rev. 56:652. 1976. Peters, H.D., B.A. Peskar, P.S. Schonhofer. Influence of prostaglandins on connective tissue cell growth and function. NaunynSchmied. Arch. Pharmacol. 297:$89. 1977. Wilkinson, D.I. Effect of vitamin A acid on the growth of keratinocytes in culture. Arch. Dermatol. Res. 263:75, 1978. Dixon-Shanies, D., J.L. Knittle. Effect of hormones on cyclic AMP levels in cultured human cells. Biochem. Biophys. Res. Com. 68:982, 1976.
Received
9/27/78
- Approved
MARCH 1979 VOL. 17 NO. 3
1/29/79
429
EFFECT OF PROSTAGLANDINS ON CYCLIC NUCLEOTIDE LEVELS IN CULTURED KERATINOCYTES
David I. Wilkinson, Ph.D. and Elaine K. Orenberg, Ph.D. Department of Dermatology Stanford University School of Medicine Stanford, California 94305 ABSTRACT Guinea pig ear epidermal cells (keratinocytes) were established in primary cultures using trypsin, and treated in their proliferative phase ofgrowth with prostaglandins El, DI, FI~, E2, D2, or F2~. This phase is induced by the addition of retinoic acid during cell plating. Intracellular content of cAMP and cGMP was measured by radioimmunoassay at various times after treatment. Maximum stimulation of cAMP levels was observed with PGD2, smaller increases with PGE2 and relatively transient rises with PGF2~ which were of low significance, but confirm earlier data. Similar results were observed with PGDI, PGEI, and PGFI~ with smaller increases. The effects of D and E PGs were biphasic. Significant increases in cGMP were immediately observed with PGD 2 and PGE2. With PGF2e, maximum cGMP levels were noted after some delay. All PGs tested showed some effect in elevating cyclic nucleotides in keratinocytes. The most striking result was the increase in cAMP on PGDz treatment. INTRODUCTION A number of studies in recent years have suggested that an elevation of cyclic adenosine -3',5'- monophosphate (cAMP) levels in mammalian epidermis takes place during the maturation (keratinization) process. Failure to achieve the necessary intracellular levels apparently may permit epidermal cells (keratinocytes) to remain in a proliferative compartment. This may be accompanied by a possible concomitant increase in cyclic guanosine -3', 5'-monophosphate (cGMP) levels. It has been suggested that the clinical expression of these conditions is the skin disease psoriasis (i). Factors which elevate epidermal cAMP levels include prostaglandins (PGs) among other compounds (2). As most of the cutaneous PG synthetase system is concentrated in the epidermis (3), it seems possible that epidermal PGs play a significant role in the maintenance of cAMP levels. Epidermis can synthesize PGs of the E,F, and
MARCH 1979 VOL. 17 NO. 3
419
PROSTAGLANDINS
D series (3,4). As a general rule, PGE species increase cAMP levels (5) and in vitro studies have shown that this also holds for human (6) and guinea pig (7) epidermis, while PGFI~ and PGFe~ are claimed to be wholly ineffective (6) or marginally effective (7). The regulation of cGMP levels in epidermis is not understood. The effect of PGs on this parameter has not been studied. Reports have indicated that PGF species may raise cGMP levels in, for instance, bovine venous strips (8) or cultured fibroblasts (9). On the other hand, a number of studies have appeared which found that PGEI and E2 induce DNA synthesis in mouse epidermis in vitro (i0) and in human (ii) and guinea pig epidermis in vivo (12). These results are difficult to interpret, because the elevation of cAMP in cultured keratinocytes results predictably in decreased DNA synthesis and mitotic inhibition (13). Further, another report found that PGs of the E and F classes had virtually no effect on 3H-thymidine uptake by cultured human keratinocytes, even at 50 DM concentrations (14). The present study has explored the effects of various PGs on cAMP and cGMP levels of cultured guinea pig keratinocytes. In particular, information regarding the following was desired: i. The effect of PGF2~ on cGMP levels. 2. The effect of PGDI and PGD2 on cAMP levels. Data on the effects of PGD species on the epidermis are not available. METHODS Tissue Treatment: Adult albino guinea pigs, Hartley strain, were obtained from Simonsen Farms, Gilroy, California. The animals were sacrificed by exsanguination and their ears removed and depilated by a 3 min treatment with Neet R (Whitehall Laboratories, Inc,, N.Y.), then thoroughly rinsed with water and scrubbed with Septadyne R (Winthrop Laboratories, N.Y.) at 1:5 v/v dilution. The ears were then rinsed briefly with 70% alcohol and used immediately. Cell Culture: Ears were cut radially into 3 pieces and planed with a Castroviejo keratotome set at 0.2 mm. Tissue so obtained was placed in GKN solution containing trypsin (0.3%) for 18-20 min at 37°C in a humidified CO 2 incubator. The epidermal and dermal moieties were isolated and agitated in the culture medium which was McCoy's 5a (modified) containing fetal calf serum (9%), Hepes (15 mM), retinoic acid (5 ~M), gentamicin (50 ~g/ml) and chlortetracycline (50 ~g/ml), pH 7.15. Cells were suspended in this medium at a density of 1.0 x 106 cells/ml and plated in 3.5 em plastic Petri dishes (Lux Scientific) at a density of 2 x 106 cells per dish. Cells were maintained at 37°C in 5% CO 2 atmosphere in a humidified incubator. After 16 hrs, the media was aspirated and replaced by media without retinoic acid. Treatment with PGs: At 48 hours, when the keratinocytes had virtually filled the dishes, the medium was removed and replaced with medium without serum (2.0 ml/dish). Incubation was continued for 3 hours, when an additional 1 ml of serum-free medium was added con-
420
MARCH 1979 VOL. 17 NO. 3
PROSTAGLANDINS
taining one of the following: PGFI, PGE2, PGFI~, PGFz~,PGDI, PGD2 to give final concentrations of 5 ~M. PGs were dissolved in ethanol before use. Final ethanol concentration was 0.1% v/v. Control dishes received ethanol alone. The dishes were maintained in the incubator. Cyclic nucleotide determinations: At various times after PG addition (time = zero) up to 150 min, dishes were harvested in triplicate by aspirating medium, rinsing the cells with 0.9% saline at 37°C, and addition of ice-cold TCA (6%). The saline rinse and TCA addition required 5 secs. Dishes were scraped off and cells homogenized in TCA using a small teflon/glass homogenizer. The suspensions were centrifuged (i0,000 g, 20 min), the supernatant extracted 4 times with twice its volume of water-saturated ether, and then lyophilized. The residue was dissolved in buffer, and appropriate dilutions acetylated and subjected to radioimmunoassay by the method of Harper and Brooker (15). Materials for radioimmunoassay were obtained from Collaborative Research. Cyclic AMP antiserum was a gift from Dr. Gary Brooker. Prostaglandins were obtain from Dr. J. Pike, Upjohn Co. The pellet from the centrifugation was dissolved in 0.5N NaOH and DNA estimated by the method of Kissane and Robins (16). RESULTS Radioimmunoassay yalidation: Duplicate aliquots were taken from each sample for assay, and the cyclic nucleotide content of each did not usually differ by more than 5% of the mean. Internal standards of cAMP and cGMP (i0, 25 a n d 5 0 femtomoles) were added to a number of samples in each experiment. In a typical series (n=15), 100.9 ± 3.47 (SEM)% of added cAMP and 98.7 ± 1.03% of cGMP was accounted for in subsequent assays. Further validation of the assays was obtained by analysis of randomly selected samples at 2 different dilutions, and by analysis before and after treatment of samples with cyclic nucleotide phosphodiesterase (beef heart, Sigma). With the latter, all material binding to antiserum was destroyed. Recovery of both nucleotides was estimated by carrying aliquots of tritiated cAMP and cGMP through the extractive processes from cell disruption to lyophilization. This was found to be 82.0 ± 1.36% for cAMP and 82.5 ± 1.02% for cGMP (means± SEM; range 73.4-89.4%; n =27). As cGMP was assayed in the presence of cAMP, the binding of cAMP by cGMP antiserum was carefully examined, for each batch of antiserum. It was found that the latter did not react detectably with cAMP in tubes containing less than 1200 femtomoles of cAMP. With the exception of samples obtained using PGD 2 at 5-20 min, none of our samples contained more than 500 fmoles/tube (i00 fmoles in diluted samples). Furthermore, there was good agreement between diluted and undiluted samples. It was concluded that increases in cGMP could not be ascribed to excess cAMP.
MARCH 1979 VOL. 17 NO. 3
421
PROSTAGLANDIN S
Cyclic AMP: iControl values showed an immediate inital decline to somewhat lower values which commenced to rise again after about 60 min to maximum values at 90-120 min, before falling again (Fig. #i). Treatment of cells with PGEI or E2 elicited a rise to significantly high values at 5 or i0 min. M a x i m u m response was observed with PGD I or PGD 2 which provoked threefold and sevenfold increases respectively over the controls. The response to E and D PGs was biphasic. After a decrease, cAMP levels again rose to high levels at 45-75 min, before falling again to or near control values. PGFI~ and PGF2~ caused a slower, smaller rise in cAMP levels, which were not particularly significant for PGFI~ (for instance, p < 0.02 at Figure #I: Variation of cAMP ( - - O - - - - ) and cGMP 6-.-~ ..... ) levels of cultured keratinocytes with time after treatment with PGs as indicated. Control dishes of cells (cAMP, • • ; cGMP ---- • . . . . S - - - - ) did not receive PGs. Each point is the mean ± SEM of 4-6 determinations from 2 experiments. Asterisks indicate the statical significance (student "t" test) over the control value for the point in question: *** ** *
p < 0.001 0.001 < p < 0.01 0.01 < p < 0.1
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Hme (minutes)
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Table#1: S t a t i s t i c a l evaluation of significance over control values of selected points from Fig. #I for cAMP (top half of Table) and cGMP (bottom half of Table) at the times (min) indicated. Figures are p values from one-sided student " t " test, and were confirmed using Wilc0xon rank test.
Time
PGEI
5 I0 15 30 45 60
<0.001
<0.01
<0.001
io
<0.02 <0.001
<0.01
15 45 60 90 120
PGDI
PGFza
PGE2
PGD2
<0.001
<0.01 <0.001 <0,001
<0.001 <0.001
<0.01
<0.001 <0.02
<0.5
<0.001 <0,30 <0.001 <0.001 <0.02
PGF2~
<0.001 <0.2 <0.2
MARCH 1979 VOL. 17 NO. 3
<0.01 <0.01
<0.01
425
PROSTAGLANDINS
30 min). These increases due to PGF were relatively transient, returning to control values after 60 min without showing biphasic behavior. In general, PGE2, D2 and F2~ caused greater responses than those observed with PGEI, DI and FI~. Cyclic GMP: As in the case of cAMP, control values showed a fall followed by an increase to higher levels and a final decrease (Fig. #i). A rather more complex reaction to treatment with PGs was observed. PGEI and Ee produced an immediate rise to about twice control levels which fell again in about 60 min. These rises showed good statistical significance (e.g., with PGE2, p < 0.001 at i0 min). PG~I and D2 showed rises of similar magnitude. For PGDI the increase was significant at i0 min, (p < 0.01) but not at any other time point. PGD2 provoked a significant rise at i0 min, (p < 0.001) and levels in PGD2-treated cultures remained generally higher than those of the control dishes until 135 min after the start, but with only indifferent significance. PGFI~ and F2~ produced immediate responses which quickly faded but showed later rises (at 45-60 and at 120 min). The increase seen with PGFe~ showed maximum significance (p < 0.001) at 60 min. DISCUSSION The most striking finding in this study was that all six PGs tested provoked a positive response in terms of both cyclic nucleotides. These responses differed widely in degree. For cAMP, the results agree well with earlier data provided by Adachi who found that'at 5 min after treatment, PGE2 at 3 ~M increased cAMP levels in pig epidermal slices by a factor of 3.2, while PGEI was less effective (2.5-fold increase) and PGF2~ gave a 1.3-fold increase which was judged to be non-significant (17). Interestingly, Adachi also studied PGAI and PGA2 and obtained small, non-significant increases in cAMP. A more surprising result is our finding of a short-term elevation in cAMP due to PGFI~ and PGF2~. Usually these agents are regarded as ineffective, although an earlier paper from this laboratory showed that both increased cAMP accumulation in guinea pig epidermis but with poor statistical significance, p < 0.01 (7). Thus three studies have shown that PGF2~ raises epidermal c~.~ by non-significant (17), marginal (7) or significant amounts (this study)~ The overall impression is that all PGs exert positive effects on epidermal cAMP. Larger increases can be observed at higher concentration; for instance, Adachi reported an 8-fold increase with PGE 2 at 25 ~M (6). Another important finding was that keratinocytes are more sensitive to PGD2 than any other PG. This suggests that if maturation is accompanied by rising cAMP levels, a tenet which is probably true, PGD 2 may be a significant agent in the process of keratinization. Experiments are in progress to determine the effects of PGD 2 on keratinocyte growth or differentiation in culture. For reasons discussed elsewhere (3) the presence of PGD 2 and its biological effects have gone unnoticed in studies of PG-keratinocyte interactions to date.
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PROSTAGLANDINS
All PGs tested provoked some increase in cGMP levels above control levels. In the first minutes after PG addition, cAMP and cGMP levels rose together and in the same direction. This is exactly the opposite behaviour to that usually encountered when studying the fluctuations of the cyclic nucleotides in a wide variety of situations such as the progress of synchronized cells (fibroblasts) through the cell cycle (18) or the passage of fibroblasts in or out of quiescence (19), when cAMP and cGMP vary in inverse proportion. However, this unindirectional reaction to PGs has been observed in some cases; Peters has shown that PGEI causes a simultaneous rise in both cAMP and cGMP in embryonic mouse fibroblasts, peaking at i0 and 5 minutes respectively (20). Since increase in cGMP is usually associated with cellular proliferative phenomena, it is tempting to explain recent reports of in vivo epidermal DNA synthesis and mitotic behaviour as due to an increase in cGMP following injection of high local levels of PGEz (10-12). However, W e have observed a burst of 3H-thymidine uptake into DNA in cultured keratinocytes after treatment with PGE 2 but without any measurable increase in amount of DNA per dish (unpublished observation). We have not been able to demonstrate growth in cultures treated with PGs. It is noteworthy that even the control levels of cAMP and cGMP fluctuate in the same direction and in a similar manner during the 2.5 hours of these experiments. This coordinated fluctuation of cAMP and cGMP in keratinocyte cultures (not subjected to treatment with drugs or other agent) is a phenomenon which we have frequently encountered and is the subject of a continuing study. Presumably the variations are related to growth patterns of the cells at 48 hours in their life in culture. The plating efficiency in primary cultures of keratinocytes is maximized by retinoic acid which also applies a mitogenic stimulus (21). The present experiments were carried out at 48 hours after plating because confluence is almost achieved at this point, but the confluent monolayer is followed by multilayer formation, stratification and the onset of keratinization. Thus, under our conditions, at 48 hours the cell population is predominantly proliferative. Serum starvation for 3 hours did not affect the cells; the same response to PGD2 has been observed after preincubation periods of from 0 to 4 hours. It is possible that a more mature (differentiating) population may react otherwise, and species differences may occur. For instance, Harper has found that human keratinocytes growing from explant skin are apparently insensitive to PGs although PGD 1 or PGD 2 was not studied (14). The reasons for the biphasic response to PGE and PGD species are unknown. It seems unlikely that the effect may be due to heterogeneity in the keratinocyte population, because a biphasic response has been reported elsewhere with cultured fibroblasts (a homogenous population) after treatment with PGEI at 5~M (22). The stability of the added PGs was studied by adding tracer amounts
MARCH 1979 VOL. 17 NO. 3
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PROSTAGLANDINS
of aH-PGEz and 3H-PGFz~ to test dishes of cells and observing recovery after 2.5 hours, by extraction of the acidified contents and TLC as before (3). There was no detectable degradation of the tracers. ACKNOWLEDGEMENT This study was supported by N.I.H. grant number AM 15107. REFERENCES i. Voorhees, J.J., N.H. Colburn, M. Stawiski, E.A. Duell, M. Haddox, N.D. Goldberg. Imbalanced cyclic AMP and cyclic GMP levels in the rapidly dividing, incompletely differentiated epidermis of psoriasis. In: Control of Proliferation in Animal Cells (C. Clarkson and R. Baserga, eds). Cold Spring Harbor Laboratory, New York, 1974, p. 635. 2. Iizuka, H., K. Adachi, K.M. Halprin, V. Levine. Adenosine and adenine nucleotides stimulation of skin (epidermal) adenylate cyclase. Biochim. Biophys. Acta 444:695. 1976. 3. Wilkinson, D.I., J.T. Walsh. Prostaglandin biosynthesis in the epidermis and dermis of young mouse skin, and the effects of calcium and cyclic nucleotides. J. Invest. Dermatol. 68:210. 1977. 4. Kingston, W.P., M.W. Greaves. Factors affecting prostaglandin synthesis by rat skin microsomes. Prostaglandins 12:51. 1976. 5. Samuelsson, B., E. Granstrom, K. Green, M. Hamburg, S. Hammarstrom. Prostaglandins. Ann. Rev. Biochem. 44:669. 1975. 6. Adachi, K., K. Yoshikawa, K.M. Halprin, V. Levine. Prostaglandins and cyclic AMP in epidermis. Brit. J. Dermatol. 92:381. 1975. 7. Aso, K., E.K. Orenberg, E.M. Farber. Reduced epidermal cyclic AMP accumulation following prostaglandin stimulation: its possible role in the pathophysiology of psoriasis. J. Invest. Dermatol. 65:375. 1975. 8. Dunham, E.W., M.K. Haddox, N.D. Goldberg. Alteration of vein 3',5' - nucleotide concentrations during changes in contractility. Proc. Nat. Acad. Sei. U.S.A. ~:815, 1974. 9. deAsua, J.L., D. Clingan, P.S. Rudland. Initiation of cell proliferation in cultured mouse fibroblasts by PGF2~. Proc. Nat. Acad. Sci. U.S.A. 72:2724. 1975. i0. Bem, J.L., M.W. Greaves. Prostaglandin El effects on epidermal cell growth in vitro. Arch. Dermatol. Forsch. 251:35. 1974. ii. Eaglstein, W.H., G.D. Weinstein. Prostaglandin and DNA synthesis in human skin. Possible relationship to ultraviolet light effects. J. Invest. Dermatol. 64:386. 1975. 12. Bentley-Phillips, C.B., H. Paulli-Jorgensen, R. Marks. The effects of prostaglandins El and F2~ on epidermal growth. Arch. Dermatol. Res. 257:233. 1977. 13. Flaxman, B.A., R.A. Harper. In vitro analysis of the control of keratinocyte proliferation in human epidermis by physiological and pharmacologic agents, j. Invest. Dermatol. 65:52. 1975.
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14.
15.
16.
17.
18.
19. 20.
21. 22.
Harper, R.A. Effect of prostaglandins on 3H-thymidine uptake into human epidermal cells in vitro. Prostaglandins 12:1019. 1976. Harper, J.F., G. Brooker. Femtomole sensitive radioimmunoassay for cAMP and cGMP after 2'0 acetylation by acetic anhydride in aqueous solution. J. Cyclic Nucleotide Res. 1:207. 1975. Kissane, J., E. Robins. The fluorometric measurement of deoxynucleic acid in animal tissues with special reference to the central nervous system. J. Biol. Chem. 233:184. 1958. Adachi, K. Epidermal cyclic AMP system and its possible role in proliferation and differentiation. In: Biochemistry of Cutaneous Epidermal Differentiation (eds. M. Seiji, I.A. Bernstein ) Univ. Tokyo Press, 1977, p. 336. Seifert, W.E., T.S. Rudland. Cyclic nucleotides and cell growth in cultured mouse cells: correlation of changes in intracellular 3',5'-cGMP concentration with a specific phase of the cell cycle. Proc. Nat. Acad. Sci. U.S.A. 71:4920. 1974. Friedman, D.L. Role of cyclic nucleotides in cell growth and differentiation. Physiol. Rev. 56:652. 1976. Peters, H.D., B.A. Peskar, P.S. Schonhofer. Influence of prostaglandins on connective tissue cell growth and function. NaunynSchmied. Arch. Pharmacol. 297:$89. 1977. Wilkinson, D.I. Effect of vitamin A acid on the growth of keratinocytes in culture. Arch. Dermatol. Res. 263:75, 1978. Dixon-Shanies, D., J.L. Knittle. Effect of hormones on cyclic AMP levels in cultured human cells. Biochem. Biophys. Res. Com. 68:982, 1976.
Received
9/27/78
- Approved
MARCH 1979 VOL. 17 NO. 3
1/29/79
429