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Endogenous prostaglandin E2 modulates calcium-induced differentiation in human skin keratinocytes
I
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PROSTAGLANDINSLEUKOTRIENES ANDESSENTIALFATTYACIDS
Endogenous Prostaglandin E2 Modulates Calcium-Induced in Human Skin Keratinocytes
Differentiation
C. B. Evans, S. Pillai and M. E. Goldyne Depurtments of Dermatology und Medicine, University qj Califor-niu, San Francisco. Frarlcisco, CA. 94121, USA (Reprint requests to MEG)
VA Medical C’cntw. Sal1
ABSTRACT. The concentration of extracellular calcium appears critical to the initiation of keratinocyte differentiation. Prostaglandins (PCs) have also been implicated in cell differentiation. Consequently, the participation of endogenous eicosanoids in calcium-induced differentiation of human keratinocytes was evaluated in vitro. Our results demonstrate that: (1) exogenously introduced PGEz, the major keratinocyte-derived eicosanoid, but not prostaglandin Iz (PGI,) or its stable metabolite 6-keto-PGF,,, enhances calcium-induced cornified envelope formation, an established marker of keratinocyte differentiation; (2) increasing extracellular calcium increased endogenous PGE2 synthesis by cultured keratinocytes; (3) blocking endogenous PGEz synthesis with indomethacin significantly suppresses calcium-induced formation of the cornified envelope; and (4) adding back PGE, to indomethacin-treated keratinocytes is able to re-establish the control level of cornified envelope formation following stimulation by calcium. These data document the participation of endogenously generated PGEz in the modulation of calcium-induced differentiation by human keratinocytes.
INTRODUCTION
Analogs of prostaglandin I, (PGI,) have also been observed to affect keratinocyte differentiation ( 14). In contrast. retinoids that inhibit keratinocyte differentiation suppress the synthesis of PCs (15). One study directly correlates the degree of keratinocyte differentiation with the ability to generate PCs ( 16). Taken together. these studies suggest a connection between extracellular calcium and endogenous PG synthesis in the induction of keratinocyte differentiation. Consequently, we studied the involvement of PGE, in the calcium-induced formation of the cornified enveloped by cultured human keratinocytes.
The extracellular concentration of calcium is crucial for the induction of differentiation among cultured skin lieratinocytes ( l-4). Low extracellular calcium (0.030.09 mM) allows preconfluent keratinocytes to proliferate but not to significantly differentiate, whereas raising extracellular calcium above 0.10 mM induces both the expression of enzymatic activity (e.g. transglutaminase) and the appearance of proteins (e.g. involucrin, filaggrin) involved in the generation of the cornified envelope, a recognized marker of the differentiated keratinocyte (5. 6). In vivo relevance of calcium is indicated by the presence of an intercellular calcium gradient in human epidermis, with higher concentrations in the region of the more differentiated keratinocytes (7, 8). How calcium induces keratinocyte differentiation is the focus of much current research. Some studies implicate a role for eicosanoids. For example, agents that induce keratinocyte differentiation, such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1,25-dihydroxyvitamin D? also induce prostaglandin (PG) synthesis (9,--12). Furthermore, the ability of TPA to induce keratinocyte differentiation and PG synthesis in mouse skm is dependent on extracellular calcium levels (13).
MATERIALS
AND METHODS
Reagents Stock solutions of indomethacin (Sigma Chemical Co.. St Louis, MO) and PGE, (Cayman Chemical Co., Ann Arbor, MI) were prepared in 95% ethanol and appropriately diluted in fresh media to achieve a final ethanol concentration below 0.1%. Aliquots of stock solutions of 1 mM 6-keto-PGF,, and 1 mM PGI, (Advanced Magnetics, Inc., Cambridge. MA) in ethylacetate were evaporated under nitrogen. reconstituted with Tris buffer (pH 9.0) containing 1% bovine serum albumin (BSA) and then aliquots added to the cell cultures to achieve a final concentration of 1 pM. PGI, was always prepared 717
778
Prostaglandins
Leukotrienes
and Essential Fatty Acids
just prior to use due to its relative instability in aqueous media. Calcium chloride was dissolved in calcium, magnesium-free Hanks Buffered Saline Solution (HBSS, Irvine Scientific, Irvine, CA), and added to the culture medium to achieve the appropriate final concentrations of calcium required in the different experimental protocols.
Keratinocyte cultures Human keratinocytes were isolated from neonatal foreskins by a 24 h treatment with 0.25% trypsin at 4°C. Primary cultures were established in serum-free keratinocyte growth medium (KGM, Clonetics Corp., San Diego, CA) containing either 0.03 or 0.15 mM calcium (depending on the goal of the particular experiment) and 70 @ml of bovine pituitary extract. At 80% confluence, cells were trypsinized and plated in flatbottom multiwell plates (6 wells/plate, Coming Inc., Coming, NY) at a density of 5 x l@ cells per well. Media was replaced every 2 days until these second passage cells reached approximately 85% confluence, at which time the described experiments were conducted.
Measurement of PGE2 A competitive enzyme immunoassay (EIA, Cayman Chemical, Ann Arbor, MI) was used to measure PGE, levels in the culture media of treated keratinocytes (17). The lower limit of detection in this assay is 3 pg/ml. Competition studies showed a 9.3% cross-reactivity with 15-keto-PGE,, 5% with PGE,, and less than 0.1% with all other major PGs and thromboxane BZ. All experimental samples were assayed in duplicate. Aliquots of media with or without the various additives were also assayed in order to subtract any background activity.
washed extensively on 1Opm filters by vaccuum filtration. The amount of radioactivity retained on the filters (i.e. incorporated into the insoluble protein that constitutes the comified envelope) was quantitated using a Beckman LS 100 scintillation counter.
RESULTS Exogenously-added PGE2 enhances cornified envelope formation For these studies, keratinocytes were grown in KGM medium containing 0.15 mM calcium, a concentration previously shown by one of the authors to be optimal for both the proliferation and differentiation of human keratinocytes (2). At approximately 60% confluence, cells were incubated in triplicate for each condition with control medium alone, and with medium containing either 10m8M PGE2, 10” M PGI,, or lo4 M 6-ketoPGF,,. The cultures received new media containing freshly prepared additives every 2 days. At 95% confluence, 10 pCi/ml of 35S-methionine were added to the media. 24 h later, incorporation of labeled methionine into the insoluble protein of the developing comified envelope was assessed as was total cellular DNA content. Figure 1 graphs comified envelope formation in response to the various PGs. Values are expressed as c.p.m. of radioactivity incorporated into SDS-insoluble, cross-linked protein per microgram of total cellular DNA. lo-8 M PGE, significantly increased comified envelope formation (p < 0.01; Student’s one-tailed t-test
400 Measurement of DNA Total cell DNA content was assessed by the method of Labarca & Paigen (18) using the DNA-specific fluorochrome bisbenzimidizole (Sigma Chemical Co., St Louis, MO). Samples were quantitated on a PerkinElmer Fluorescence Spectrophotometer-204 with reference to known quantities of DNA standards using an excitation wavelength of 356 nm and an emission wavelength of 458 nm. All samples were assayed in duplicate.
Measurement of cornified envelope formation Rate of formation of comified envelopes (19) was assessed by incubating the appropriate cultures with 10 pCi/ml of 35S-L-methionine (Amersham, Arlington Heights, IL). The 35S-methionine-labeled cells were then harvested at the appropriate times (depending on the experiment) using 2% SDS-20 mM DTT and the proteins solubilized by boiling for at least 30 min. The SDSinsoluble, labeled, cross-linked envelope proteins were
300
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ze
200
0
100 0
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a
Fig. 1 Effects of PC&&, PGI,, and 6-keto-PGF,, on corniced envelope formation. Keratinocytes (60% confluent) were exposed to: (a) = control media alone (KGM, 0.15mM calcium); (b) = 1W M PGE,; (c) = lo-6 M PGI,; (d) = l@ M 6-keto-PGF,,. At 95% confluence 10 pCi/ml of ?S-methionine were added to the cultures for the last 24 h of the incubation. Cells were harvested and analyzed as described in materials and methods. Values are expressed as the mean + SEM (n = 3) c.p.m/pg DNA. The asterisk indicates a value statistically greater than the control (p < 0.01; one-tailed Student’s t-test).
Endogenous for independent sample means) whereas lad M concentrations of either 6-keto-PGF,, or PGI, failed to significantly affect comified envelope formation when compared to the control.
Calcium
increases endogenous
PGE2 production
The ability of exogenous PGE? to enhance comified envelope formation raised the question of whether increasing extracellular calcium would in fact induce the endogenous generation of PGE, by cultured human keratinocytes. For these studies, keratinocytes were first grown in low calcium (0.03 mM) KGM. At approximately 85% contluence, replicate wells (n = 3) received fresh medium containing 1.2 mM calcium whereas control wells received fresh medium containing 0.03 mM calcium. After 1 h and 48 h, media were collected and analyzed for PGE, by EIA. Figure 2 compares the 1 and 48 h levels of PGE, present in the keratinocyte supematants containing 1.2 mM calcium to the control supematants containing 0.03 mM calcium. Values are expressed as pg/pg DNA to account for changes in cell number over 48 h. Mean PGEl levels were already higher at 1 h in response to 1.2 mM calcium compared to control levels (p < 0.005; Student’s one-tailed t-test for independent sample means). At 48 h, there was an even greater difference between the mean PGE, levels in the high versus low calcium cultures (p < 0.0005). 1W M indomethacin prevented the increase in PGE2 levels in response to calcium, verifying that the detected reactivity seen was not artifact (data not shown); indomethacin alone did not interfere with the assay.
T!
Indomethacin suppresses envelope formation
Prostaglandin
calcium-induced
E?
779
cornified
To further implicate PGE2 in the calcium-induced generation of comified envelope, studies were done to determine whether indomethacin, an inhibitor of cyclooxygenase, should suppress calcium-induced comified envelope formation. Keratinocytes. grown in KGM containing 0.03 mM calcium until 85% confluent, were subsequently exposed to 1.2 mM calcium plus 3 @/ml j5S-methionine for 24 h in the absence or presence of 10” M indomethacin. Labeled methionine incorporation and total cellular DNA were then assessed as in the previous studies. Figure 3 shows that comified envelope formation in response to increased extracellular calcium was significantly suppressed by indomethacin (p < 0.05; Student’s one-tailed t-test for independent sample means).
Adding back PGEz to indomethacin-treated keratinocytes restores cornified envelope formation to control levels Because the effects of indomethacin on cell function could potentially involve more than just the inhibition of PG generation, the study summarized in Figure 3 included the adding back of 10m7M PGE, to indomethacin-treated keratinocytes grown in 1.2 mM calcium. Figure 3 shows that exogenous PGE, was able to restore comified envelope formation to the control level in the presence of indomethacin
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a
1 hr
48 hr
Fig. 2 Effect of calcium on endogenous PG production. The media from cells incubated for 1 or 48 h in KGM containing either 0.03 mM calcium (control: solid bars) or I .2 mM calcium (hatched bars) were analyzed for PGE,. The data are expressed as mean f SEM (n = 3) pg/pg DNA. Asterisks indicate values statistically greater than the respective controls (p < 0.005 for 1 h and < O.OOOSfor 4X h; one-tailed Student’s t-test).
b
c
d
Fig. 3 Effect of indomethacin. in the absence and presence of PGE,, on calcium-induced comified envelope formation. Keratinocytes (85% confluent) were maintained either in control media (KGM, 0.03 mM calcium) or exposed to 1.2 mM calcium for 24 h in the absence or presence of IO6 M indomethacin. All cultures were co-incubated with 3 uCi/ml ‘%-methionine: (a) = control media (KGM, 0.03 mM calcium); (b) = 1.2 mM calcium : Cc)= I .2 mM calcium + IO” M indomethacin: (d) = I .2 mM calcium + I P M indomethacin + 1OF7M PGE,. Data are expressed as mean f SEM (n = 3) c.p.m/pg DNA. Asterisks indicate values statistically greater than both the control (a) and the indomethacin-treated cells in I .2 mM calcium cc) (p < 0.05: one-tailed Student’s t-test).
780
Prostaglandins
Leukotrienes
and Essential Fatty Acids
DISCUSSION Our data show that PGEl is involved in the calciuminduced generation of the comified envelope in cultured human keratinocytes. While the data do not reveal how PGE, generation is induced by calcium, Clark et al recently characterized a cytosolic phospholipase A2 (cPLA,) (20) which requires a rise in intracellular calcium levels to become activated; moreover, this cPLA, shows specificity for arachidonyl-containing phospholipids. We have, in fact, recently documented the presence of cPLA> in our cultured human skin keratinocytes (21) so that activation of this enzyme following the calcium switch would be consistent with our findings of increased PGE? synthesis. The hypothesis that PGE2 participates in human keratinocyte differentiation has been challenged. Whereas the phorbol ester, TPA, induces calciumdependent PGE, generation and differentiation in murine keratinocytes (13), DeLeo et al have claimed that a similar induction of arachidonic acid metabolism by TPA in human keratinocytes does not occur although terminal differentiation is still induced (22). Their studies, however, involved prelabeling of cells with “H-arachidonic acid and monitored only 3H-PG release. Neither the purity of the ‘H-arachidonic acid nor the tritium exchange possibly occurring during labeling were assessed. Because of these potential pitfalls, we recently examined endogenous PGE, release from human keratinocytes in response to TPA using the enzyme immunoassay employed in the present studies (23). We observed a dosedependent increase in PGEz generation by human keratinocytes in response to TPA (g-fold at 3 h and 12fold at 24 h with l@* g/ml of TPA). Therefore we feel the correlation between differentiation agonists and their ability to stimulate PGE, production ( 10-l 2) applies to human keratinocytes and that further exploration into the mechanism through which PGE, modulates human keratinocyte differentiation is justified. The ability of PGEl to enhance human keratinocyte differentiation is compatable with the earlier observations of Kirscher who found that PGE, enhanced maturation of chick skin and produced precocious keratinization (24). It is also well recognized that essential fatty acid deficiency alters keratinocyte differentiation(25, 26). In this context, Ziboh et al (25) observed that application of PGE, to the skin of EFA-deficient rats caused a gross and histologic normalization of the previously hyperkeratotic skin. This is not to assert that PGE, is responsible for regulating the total process of differentiation; Elias et al (26) in fact showed that topical PGE, application does not restore altered barrier function in EFA-deficient skin. At the same time, recent investigations suggest that PGEz (and possibly other arachidonic acid metabolites) participate in the differentiation of cells other than keratinocytes (27, 28). Therefore, our data. in conjunction with the previous studies cited, offer strong support for PGE? as an autocrine participant
in the complex process of keratinocyte differentiation. Identifying the scope of PGE, involvement will require further investigation.
Acknowledgments We thank MS Shyamala Sundemath and Mr Mark Bogen for their expert assistance in keratinocyte culturing techniques. This paper was supported by the Merit Review Grant from Department of Veterans Affairs (MEG).
References 1. Hennings H. Michael D, Cheng C. Steinert P, Holbrook K, Yuspa S H. Calcium regulation of growth and differentiation of mouse epidermal cells in culture. Cell 1980: 19: 245-254. 2. Pillai S, Bickle D. Hincenbergs M, Elias P. Biochemical and morphological characterization of growth and differentiation of normal human neonatal keratinocytes in a serum-free media. J Cell Phys 1988: 134: 229-237. 3. Yuspa S H, Kilkenny A E, Steinert P M. Roop D R. Expression of murine epidermal differentiation markers is tightly regulated by restricted extracellular calcium concentrations in vitro. J Cell Biol 1989; 109: 1207- 12 17. 4. Pillai S, Bickle D. Mancianti M L, Cline P. Hincenbergs M. Calcium regulation of growth and differentiation of normal human keratinocytes: Modulation of differentiation competence by stages of growth and extracellular calcium. J Cell Phys 1990: 143: 294-302. 5. Sun T T, Green H. Differentiation of the epidermai keratinocyte in cell culture: Formation of the comified envelope. Cell 1976: 9: 511-571. 6. Hohl, D. Comified cell envelope. Demratologica 1990: 180: 201-21 I. 7. Menon G K. Elias P M. Ultrastructural localization of calcium in psoriatic and normal human epidermis. Arch Dermatol 1991; 127: 57-63. 8. Menon G K. Grayson S. Elias P M. Ionic calcium reservoirs in mammalian epidermis: ultrastructural localization by ion-capture cytochemistry. J Invest Dermatol 1985: 84: 508-512. 9. Lichti U, Yuspa S H. Modulation of tissue and epidermal transglutaminases in mouse epidermal cells after trearment with TPA and/or retinoic acid in vivo and in culture. Cancer Res 1988: 48: 74-81. 10. Pillai S. Bickle D. Role of intracellular free calcium in the comified envelope formation of keratinocytes: Differences in the mode of action of extracellular calcium and 1,25-dihydroxy Vitamin D,. J Cell Phys 1991: 146: 94-100. 11. Furstenberger G. Marks F. Early prostaglandin E synthesis is an obligatory event in the induction of cell proliferation in mouse epidermis in vivo by the phorbol ester TPA. Biochem Biophys Res Commun 1980: 92: 749-756. 12. Wakasugi M. Noguchi T. moue M et al. Vitamin Di stimulates the production of prostacyclin by vascular smooth muscle cells. Prostaglandins 1991: 42(2): 127-136. 13. Ganss M. Seemann D, Furstenberger G, Marks F. Calcium-dependent release of arachidonic acid from a murine epidermal cell line induced by the tumor promoter TPA or ionophore A23187. FEBS Letters 1982; 142( 1): 54-58. 14. Baden H P, Goldaber M L. Kvedar J C. Keratinocytes stimulate prostaglandin I2 synthesis by 3T3 cells and exhibit enhanced comification when exposed to prostaglandin I2 analogues. J Cell Phys 1992: 150: 269-275. 15. Elattar T M A, Lin H S. Effect of retinoids and carotenoids on prostaglandin formation by oral squamous carcinoma cells. Prostaglandins Leukot Essen Fatty Acids 1991; 43: 175-178.
Endogenous 16. Cameron G S, Baldwin J K, Jasheway D W, Patrick K E, Fisher S M. Arachidonic acid metabolism varies with the state of differentiation in density gradient separated mouse epidermal cells. J Invest Dermatol 1990: Y4(3): 292-396. 17. Pradellea I’. Grassi J. and Maclouf J. Enzyme immunoa\\ays of eicosanoids using acetylcholine esterase as label: an alternative to radioimmunoassay. Anal Biochem 19X5: 57: 1170-I 173. IX. Labarca C. Paigen K. A simple. rapid and sensitive DNA assay procedure. Anal Biochem 1980: 102: 34&352. 19. King 1. Mella S L. Sartorelli A L. A sensitive method to quantify the terminal differentiation of cultured epidermal cells. Exp Cell Res 1986: 167: 252-2.56. 20. Clark J D, Lin L L. Krir R W et al. A novel arachidonic acid-selective cytosolic PLAZ contains a calciumdependent translocation domain with homology to PKC and GAP. Cell 1991; 65: 1043-1051. 21. Goldyne M E, Evans C B. Clark J D. Demonstration of The cytosolic phospholipase A2 in human skin keratinocytes and fibroblasts. 1993: submitted to Prostaglandins. 71. DeLeo V .4, Scheide S, Horlich H et al. The effect of 12.O-tetradeca-noylphorbol-1%acetate (TPA) on
23.
24.
2s.
26.
27.
28.
Prostaglandin
E,
phospholipid metabolism of human epidennal keratinocytes. Carcinogenesis 1986: 7: 937-932. Goldyne M E. Evans C B. I?-O-tetradecanoylphorbol13acetate and the induction of prostaglandin E2 penemtion by human keratinocytes: A re-evaluation. Submilted to Carcinogenesis. 1993. Kischer C W. Effect of specific prostaglandins on development of chick embryo skin and down feather organ in vitro. Dev Biol 1967: 16: 203-215. Ziboh V A, Hsia S L. Effects of prostaglandin E7 on rat skin: inhibition of sterol ester biosynthesis and clearing o! scaly lesion5 in essential fatty acid deficiency. J Lipid Res 1972; 13: 458467. Elias P M. Brown B. Ziboh V A. The permeability harrier in essential fatty acid deficiency: evidence for a direct role for linoleic acid m barrier function. J Invest Dermatol 1980: 74: 230-233. Koehler L. Hass R, Wessel K et al. Altered arachldonic acid metabolism during differentiation of the human monoblastoid cell line LJ937. Biochem Biophy\ Acta 1990: 1042: 395403. Hume R, Kelly R. Cossar D et al. Self-differentiation of human fetal lung organ culture: the role of prostaglandins PGE, and PGF,,. Exp Cell Rea 1991: 19-I: I II -1 I:.
7X
~
PROSTAGLANDINSLEUKOTRIENES ANDESSENTIALFATTYACIDS
Endogenous Prostaglandin E2 Modulates Calcium-Induced in Human Skin Keratinocytes
Differentiation
C. B. Evans, S. Pillai and M. E. Goldyne Depurtments of Dermatology und Medicine, University qj Califor-niu, San Francisco. Frarlcisco, CA. 94121, USA (Reprint requests to MEG)
VA Medical C’cntw. Sal1
ABSTRACT. The concentration of extracellular calcium appears critical to the initiation of keratinocyte differentiation. Prostaglandins (PCs) have also been implicated in cell differentiation. Consequently, the participation of endogenous eicosanoids in calcium-induced differentiation of human keratinocytes was evaluated in vitro. Our results demonstrate that: (1) exogenously introduced PGEz, the major keratinocyte-derived eicosanoid, but not prostaglandin Iz (PGI,) or its stable metabolite 6-keto-PGF,,, enhances calcium-induced cornified envelope formation, an established marker of keratinocyte differentiation; (2) increasing extracellular calcium increased endogenous PGE2 synthesis by cultured keratinocytes; (3) blocking endogenous PGEz synthesis with indomethacin significantly suppresses calcium-induced formation of the cornified envelope; and (4) adding back PGE, to indomethacin-treated keratinocytes is able to re-establish the control level of cornified envelope formation following stimulation by calcium. These data document the participation of endogenously generated PGEz in the modulation of calcium-induced differentiation by human keratinocytes.
INTRODUCTION
Analogs of prostaglandin I, (PGI,) have also been observed to affect keratinocyte differentiation ( 14). In contrast. retinoids that inhibit keratinocyte differentiation suppress the synthesis of PCs (15). One study directly correlates the degree of keratinocyte differentiation with the ability to generate PCs ( 16). Taken together. these studies suggest a connection between extracellular calcium and endogenous PG synthesis in the induction of keratinocyte differentiation. Consequently, we studied the involvement of PGE, in the calcium-induced formation of the cornified enveloped by cultured human keratinocytes.
The extracellular concentration of calcium is crucial for the induction of differentiation among cultured skin lieratinocytes ( l-4). Low extracellular calcium (0.030.09 mM) allows preconfluent keratinocytes to proliferate but not to significantly differentiate, whereas raising extracellular calcium above 0.10 mM induces both the expression of enzymatic activity (e.g. transglutaminase) and the appearance of proteins (e.g. involucrin, filaggrin) involved in the generation of the cornified envelope, a recognized marker of the differentiated keratinocyte (5. 6). In vivo relevance of calcium is indicated by the presence of an intercellular calcium gradient in human epidermis, with higher concentrations in the region of the more differentiated keratinocytes (7, 8). How calcium induces keratinocyte differentiation is the focus of much current research. Some studies implicate a role for eicosanoids. For example, agents that induce keratinocyte differentiation, such as 12-O-tetradecanoylphorbol-13-acetate (TPA) and 1,25-dihydroxyvitamin D? also induce prostaglandin (PG) synthesis (9,--12). Furthermore, the ability of TPA to induce keratinocyte differentiation and PG synthesis in mouse skm is dependent on extracellular calcium levels (13).
MATERIALS
AND METHODS
Reagents Stock solutions of indomethacin (Sigma Chemical Co.. St Louis, MO) and PGE, (Cayman Chemical Co., Ann Arbor, MI) were prepared in 95% ethanol and appropriately diluted in fresh media to achieve a final ethanol concentration below 0.1%. Aliquots of stock solutions of 1 mM 6-keto-PGF,, and 1 mM PGI, (Advanced Magnetics, Inc., Cambridge. MA) in ethylacetate were evaporated under nitrogen. reconstituted with Tris buffer (pH 9.0) containing 1% bovine serum albumin (BSA) and then aliquots added to the cell cultures to achieve a final concentration of 1 pM. PGI, was always prepared 717
778
Prostaglandins
Leukotrienes
and Essential Fatty Acids
just prior to use due to its relative instability in aqueous media. Calcium chloride was dissolved in calcium, magnesium-free Hanks Buffered Saline Solution (HBSS, Irvine Scientific, Irvine, CA), and added to the culture medium to achieve the appropriate final concentrations of calcium required in the different experimental protocols.
Keratinocyte cultures Human keratinocytes were isolated from neonatal foreskins by a 24 h treatment with 0.25% trypsin at 4°C. Primary cultures were established in serum-free keratinocyte growth medium (KGM, Clonetics Corp., San Diego, CA) containing either 0.03 or 0.15 mM calcium (depending on the goal of the particular experiment) and 70 @ml of bovine pituitary extract. At 80% confluence, cells were trypsinized and plated in flatbottom multiwell plates (6 wells/plate, Coming Inc., Coming, NY) at a density of 5 x l@ cells per well. Media was replaced every 2 days until these second passage cells reached approximately 85% confluence, at which time the described experiments were conducted.
Measurement of PGE2 A competitive enzyme immunoassay (EIA, Cayman Chemical, Ann Arbor, MI) was used to measure PGE, levels in the culture media of treated keratinocytes (17). The lower limit of detection in this assay is 3 pg/ml. Competition studies showed a 9.3% cross-reactivity with 15-keto-PGE,, 5% with PGE,, and less than 0.1% with all other major PGs and thromboxane BZ. All experimental samples were assayed in duplicate. Aliquots of media with or without the various additives were also assayed in order to subtract any background activity.
washed extensively on 1Opm filters by vaccuum filtration. The amount of radioactivity retained on the filters (i.e. incorporated into the insoluble protein that constitutes the comified envelope) was quantitated using a Beckman LS 100 scintillation counter.
RESULTS Exogenously-added PGE2 enhances cornified envelope formation For these studies, keratinocytes were grown in KGM medium containing 0.15 mM calcium, a concentration previously shown by one of the authors to be optimal for both the proliferation and differentiation of human keratinocytes (2). At approximately 60% confluence, cells were incubated in triplicate for each condition with control medium alone, and with medium containing either 10m8M PGE2, 10” M PGI,, or lo4 M 6-ketoPGF,,. The cultures received new media containing freshly prepared additives every 2 days. At 95% confluence, 10 pCi/ml of 35S-methionine were added to the media. 24 h later, incorporation of labeled methionine into the insoluble protein of the developing comified envelope was assessed as was total cellular DNA content. Figure 1 graphs comified envelope formation in response to the various PGs. Values are expressed as c.p.m. of radioactivity incorporated into SDS-insoluble, cross-linked protein per microgram of total cellular DNA. lo-8 M PGE, significantly increased comified envelope formation (p < 0.01; Student’s one-tailed t-test
400 Measurement of DNA Total cell DNA content was assessed by the method of Labarca & Paigen (18) using the DNA-specific fluorochrome bisbenzimidizole (Sigma Chemical Co., St Louis, MO). Samples were quantitated on a PerkinElmer Fluorescence Spectrophotometer-204 with reference to known quantities of DNA standards using an excitation wavelength of 356 nm and an emission wavelength of 458 nm. All samples were assayed in duplicate.
Measurement of cornified envelope formation Rate of formation of comified envelopes (19) was assessed by incubating the appropriate cultures with 10 pCi/ml of 35S-L-methionine (Amersham, Arlington Heights, IL). The 35S-methionine-labeled cells were then harvested at the appropriate times (depending on the experiment) using 2% SDS-20 mM DTT and the proteins solubilized by boiling for at least 30 min. The SDSinsoluble, labeled, cross-linked envelope proteins were
300
z!
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200
0
100 0
-c
a
Fig. 1 Effects of PC&&, PGI,, and 6-keto-PGF,, on corniced envelope formation. Keratinocytes (60% confluent) were exposed to: (a) = control media alone (KGM, 0.15mM calcium); (b) = 1W M PGE,; (c) = lo-6 M PGI,; (d) = l@ M 6-keto-PGF,,. At 95% confluence 10 pCi/ml of ?S-methionine were added to the cultures for the last 24 h of the incubation. Cells were harvested and analyzed as described in materials and methods. Values are expressed as the mean + SEM (n = 3) c.p.m/pg DNA. The asterisk indicates a value statistically greater than the control (p < 0.01; one-tailed Student’s t-test).
Endogenous for independent sample means) whereas lad M concentrations of either 6-keto-PGF,, or PGI, failed to significantly affect comified envelope formation when compared to the control.
Calcium
increases endogenous
PGE2 production
The ability of exogenous PGE? to enhance comified envelope formation raised the question of whether increasing extracellular calcium would in fact induce the endogenous generation of PGE, by cultured human keratinocytes. For these studies, keratinocytes were first grown in low calcium (0.03 mM) KGM. At approximately 85% contluence, replicate wells (n = 3) received fresh medium containing 1.2 mM calcium whereas control wells received fresh medium containing 0.03 mM calcium. After 1 h and 48 h, media were collected and analyzed for PGE, by EIA. Figure 2 compares the 1 and 48 h levels of PGE, present in the keratinocyte supematants containing 1.2 mM calcium to the control supematants containing 0.03 mM calcium. Values are expressed as pg/pg DNA to account for changes in cell number over 48 h. Mean PGEl levels were already higher at 1 h in response to 1.2 mM calcium compared to control levels (p < 0.005; Student’s one-tailed t-test for independent sample means). At 48 h, there was an even greater difference between the mean PGE, levels in the high versus low calcium cultures (p < 0.0005). 1W M indomethacin prevented the increase in PGE2 levels in response to calcium, verifying that the detected reactivity seen was not artifact (data not shown); indomethacin alone did not interfere with the assay.
T!
Indomethacin suppresses envelope formation
Prostaglandin
calcium-induced
E?
779
cornified
To further implicate PGE2 in the calcium-induced generation of comified envelope, studies were done to determine whether indomethacin, an inhibitor of cyclooxygenase, should suppress calcium-induced comified envelope formation. Keratinocytes. grown in KGM containing 0.03 mM calcium until 85% confluent, were subsequently exposed to 1.2 mM calcium plus 3 @/ml j5S-methionine for 24 h in the absence or presence of 10” M indomethacin. Labeled methionine incorporation and total cellular DNA were then assessed as in the previous studies. Figure 3 shows that comified envelope formation in response to increased extracellular calcium was significantly suppressed by indomethacin (p < 0.05; Student’s one-tailed t-test for independent sample means).
Adding back PGEz to indomethacin-treated keratinocytes restores cornified envelope formation to control levels Because the effects of indomethacin on cell function could potentially involve more than just the inhibition of PG generation, the study summarized in Figure 3 included the adding back of 10m7M PGE, to indomethacin-treated keratinocytes grown in 1.2 mM calcium. Figure 3 shows that exogenous PGE, was able to restore comified envelope formation to the control level in the presence of indomethacin
6004
20-
2 N i Y
I0
a
1 hr
48 hr
Fig. 2 Effect of calcium on endogenous PG production. The media from cells incubated for 1 or 48 h in KGM containing either 0.03 mM calcium (control: solid bars) or I .2 mM calcium (hatched bars) were analyzed for PGE,. The data are expressed as mean f SEM (n = 3) pg/pg DNA. Asterisks indicate values statistically greater than the respective controls (p < 0.005 for 1 h and < O.OOOSfor 4X h; one-tailed Student’s t-test).
b
c
d
Fig. 3 Effect of indomethacin. in the absence and presence of PGE,, on calcium-induced comified envelope formation. Keratinocytes (85% confluent) were maintained either in control media (KGM, 0.03 mM calcium) or exposed to 1.2 mM calcium for 24 h in the absence or presence of IO6 M indomethacin. All cultures were co-incubated with 3 uCi/ml ‘%-methionine: (a) = control media (KGM, 0.03 mM calcium); (b) = 1.2 mM calcium : Cc)= I .2 mM calcium + IO” M indomethacin: (d) = I .2 mM calcium + I P M indomethacin + 1OF7M PGE,. Data are expressed as mean f SEM (n = 3) c.p.m/pg DNA. Asterisks indicate values statistically greater than both the control (a) and the indomethacin-treated cells in I .2 mM calcium cc) (p < 0.05: one-tailed Student’s t-test).
780
Prostaglandins
Leukotrienes
and Essential Fatty Acids
DISCUSSION Our data show that PGEl is involved in the calciuminduced generation of the comified envelope in cultured human keratinocytes. While the data do not reveal how PGE, generation is induced by calcium, Clark et al recently characterized a cytosolic phospholipase A2 (cPLA,) (20) which requires a rise in intracellular calcium levels to become activated; moreover, this cPLA, shows specificity for arachidonyl-containing phospholipids. We have, in fact, recently documented the presence of cPLA> in our cultured human skin keratinocytes (21) so that activation of this enzyme following the calcium switch would be consistent with our findings of increased PGE? synthesis. The hypothesis that PGE2 participates in human keratinocyte differentiation has been challenged. Whereas the phorbol ester, TPA, induces calciumdependent PGE, generation and differentiation in murine keratinocytes (13), DeLeo et al have claimed that a similar induction of arachidonic acid metabolism by TPA in human keratinocytes does not occur although terminal differentiation is still induced (22). Their studies, however, involved prelabeling of cells with “H-arachidonic acid and monitored only 3H-PG release. Neither the purity of the ‘H-arachidonic acid nor the tritium exchange possibly occurring during labeling were assessed. Because of these potential pitfalls, we recently examined endogenous PGE, release from human keratinocytes in response to TPA using the enzyme immunoassay employed in the present studies (23). We observed a dosedependent increase in PGEz generation by human keratinocytes in response to TPA (g-fold at 3 h and 12fold at 24 h with l@* g/ml of TPA). Therefore we feel the correlation between differentiation agonists and their ability to stimulate PGE, production ( 10-l 2) applies to human keratinocytes and that further exploration into the mechanism through which PGE, modulates human keratinocyte differentiation is justified. The ability of PGEl to enhance human keratinocyte differentiation is compatable with the earlier observations of Kirscher who found that PGE, enhanced maturation of chick skin and produced precocious keratinization (24). It is also well recognized that essential fatty acid deficiency alters keratinocyte differentiation(25, 26). In this context, Ziboh et al (25) observed that application of PGE, to the skin of EFA-deficient rats caused a gross and histologic normalization of the previously hyperkeratotic skin. This is not to assert that PGE, is responsible for regulating the total process of differentiation; Elias et al (26) in fact showed that topical PGE, application does not restore altered barrier function in EFA-deficient skin. At the same time, recent investigations suggest that PGEz (and possibly other arachidonic acid metabolites) participate in the differentiation of cells other than keratinocytes (27, 28). Therefore, our data. in conjunction with the previous studies cited, offer strong support for PGE? as an autocrine participant
in the complex process of keratinocyte differentiation. Identifying the scope of PGE, involvement will require further investigation.
Acknowledgments We thank MS Shyamala Sundemath and Mr Mark Bogen for their expert assistance in keratinocyte culturing techniques. This paper was supported by the Merit Review Grant from Department of Veterans Affairs (MEG).
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