Novel regulatory actions of 1α,25-dihydroxyvitamin D3 on the metabolism of polyphosphoinositides in murine epidermal keratinocytes

JOURNAL OF CELLULAR PHYSIOLOGY 132:131-136 (1987)

Novel Regulatory Di h yd roxyvi tam i n D3

Actions of 1 a,25- on the Metabolism of

Pol yphosphoi nosit ides in Muri ne Epidermal Keratinocytes

WILSON TANG, VINCENT A. ZIBOH,* R. RIVKAH ISSEROFF, AND D. MARTINEZ Department of Dermafoiogy, School of Medicine, University of California,

Davis, California 95616

The in vitro incubation of mur ine keratinocytes in the presence of 1a,25- dihydroxyvitamin D3 enhanced t h e rapid hydrolysis of the prelabeled keratinocyte polyphosphoinositides (polyPtdlns) when compared to untreated cells. The rapid hydrolysis of the polyPtdlns and the release of the inositol phos- phosphates (particularly InsP3 and InsP2) precede the onset of differentiation of these cells. These data therefore suggest that la,25-didydroxyvitamin D3 functions in vitro to initiate the rapid generation of InsP3 from cellular poly- Ptdlns; this in tu rn may mobilize intracellular Ca2+, thus providing the signal which program the murine keratinocytes from a proliferating mode into a differentiating mode.

Although vitamin D3 is synthesized by skin from en- dogenous 7-dehydrocholesterol (MacLaughlin et al., 1982), it is the resulting kidney metabolite, 1 a,25-dihydroxyvitamin D3 [la,25(OH)zD3] which is known to possess hormonal regulatory activities in various target organs via a receptor-mediated mechanism (DeLuca and Schnoes, 1976). Receptors for ~ X , ~ ~ ( O H ) ~ D ~ have recently been reported in fresh and cultured human skin (Feldman et al., 19801, in cytosol prepared from rat (Simpson and DeLuca, 1980), and in nuclear and cytosol fractions of whole mouse skin in vivo and in vitro (Cle- mens et al., 1981), suggesting that this tissue, along with bone and intestine, is an additional target organ for this hormone. In fact, it was reported recently that the concentration of la,%(oH)zD3 binding macromole- cule in mouse skin chromatin increased significantly during fetal and neonatal development (Horiuchi et al., 1985), thus prompting the suggestion that increase in this vitamin-chromatin binding may play a regulatory role in epidermal proliferation and differentiation during skin development. The mechanism for such a possible role is, however, unclear. Interestingly, the la,25(OH)zD3 was recently reported to enhance the differentiation of mouse keratinocytes in culture (Hosomi et al., 1983). Such a capability of la,25(OH)zD3 to induce cellular differentiation in various cell lines has also been reported in human (Miyaura et al., 1981) and mouse (Abe et al., 1981) myeloid leukemia cells.

Despite these known modulatory effects of la,25 (OH)zD3 on proliferation and differentiation, the biochemical mechanisms associated with these events are still unclear. Since a recent study from our laboratory has demonstrated that a close relationship exists between the hydrolysis of phosphatidylinositol-4,5-bisphosphate (PtdIns4,5P2), the generation of inositol-1,4,5,-

0 1987 ALAN R. LISS, INC.

triphosphate (InsPs), and differentiation in normal murine keratinocyte, we were prompted to test whether or not the exogenous addition of 1a,25(OH)zD3 (a vitamin D3 metabolite reported to possess receptors in keratinocytes) to keratinocytes in culture enhances the hydrolysis of the cellular polyPtdIns and the generation of inositol phosphates (second messengers); and if so, whether the generation of InsP3 (in particular) precedes or correlates with the onset of the murine keratinocyte differentiation in culture.

MATERIALS AND METHODS Cell culture conditions

Primary keratinocyte cultures were prepared from mouse epidermis according to the procedure of Yuspa and Harris (1974). Briefly, single cell suspension was obtained through overnight trypsinization of the epidermis at 4°C with 0.25% trypsin in Hank’s balanced salt solution. Isolated celIs (6 x 106/60 mm plate) were plated and labeled overnight with 0.4 pCi of my~-[U-~~C]-inosi- to1 (53.3 mCi/mmol, Amersham, Arlington Heights, IL) in Dulbecco’s Modified Eagles’ Medium (DMEM) (Flow Laboratories, Inglewood, VA) supplemented with 10% fetal calf serum (FCS) (Irvine Scientific, Irvine, CA) at 37°C with 5% C02.

Abbreviations

PtdIns, Phosphatidylinositol PtdIns4P, Phosphatidylinositol 4- phosphate PtdIns4,5Pz, Phosphatidylinositol,4,5-bisphosphate InsP, Inositol phosphate InsPz, Inositol bisphosphate InsPs, Inositol triphosphate lrr,25(OH)2Ds, la,25dihydroxyvitamin D3

Received August 25,1986; accepted December 22, 1986.

*To whom reprint requests/correspondence should be addressed.

132 TANG, ZIBOH, ISSEROFF. AND MARTINEZ

Incubation of la,25(OH)zD3 with primary keratinocytes in culture

In preliminary experiments, varying concentrations of exogenous lar,25(OH)&(12 x M to 12 x M) previously dissolved in ethanol were added to the medium containing cells and 14C-myo-inositol a t plating time, as described above. These initial experiments were to determine the long-term effect as well as the maximal concentration of la,25(OH)zD3 to exert a reaction on the cells. The maximal concentration of ethanol in the medium was 0.1%, a concentration previously determined not to impair activity of the cells. In control experiments the cells were plated with the same amount of ethanol and precursor ‘‘C-inositol, but containing no la,25 (OH)zD3. In subsequent experiments, lcu,%(OH)2D3 at a concentration of 12 x 10 -lo M was used to determine its effect on the incorporation of precursor 14C- inositol into the keratinocyte ptdIns. Incubations were as described above, with or without vitamin D3 metabolite as control and Liclz (10 mM).

To evaluate the effects of the la,25(OH)2D3 on the hydrolysis of the 14C-PtdIns and the generation of InsP3, primary cultures of keratinoc es (6 x 106/60-mm plate)

in DMEM supplemented with 10% FCS for 36 hr. Under our culture conditions, during the first 36 hr, the cells grew as a monolayer and were characterized by a predominant population of proliferating, morphologically “basaloid or cuboidal” cells, although this population of cells also contains a small subset of cells already entering the differentiating mode since primary cultures of keratinocytes are in a dynamic state. The population of cells at 36 hr are therefore predominantly proliferating cells with a minimal number of differentiating cells as as- sessed by elevated levels of activator (Isseroff, et al., 19831, a marker of keratinocyte differentiation. Beyond 36 hr, evidence of keratinocyte differentiation, which was characterized by the morphological appearance of clumps of keratofilaments within the cell and cornified envelope formation (squames), was discernible (Breit- kreutz et al., 1981). Thus, in this study, the 14C-containing medium was aspirated from the cultured cells at 36 hr after the initiation of incubation, followed by the washing of the cells three times with phosphate-buffered saline. Fresh medium supplemented with or without lar,25(OH)$3(12 x -l0M) in ethanol was added to the cultures. The incubation was carried out at 37°C and the reaction stopped at varying time periods (0, 30, 60, and 120 sec) to determine the extent of hydrolysis. A schematic diagram of the experimental protocol used in these studies is given (Fig. 1).

Extraction and separation of 14C- polyphosphoinositides/inositol phosphates

Cellular products containing 14C-poly-PtdIns. Cells were scraped from the petri dish in methanol with teflon spatulas into ground-glass homogenizers a t the time points described above. The homogenization medium containing the cells was made to volume with acidified CHC13/CH3OH (2:1, v/v) and homogenized. Partition of the mixture was achieved by the addition of 0.2 vol of 0.25% CaClz and centrifugation. The organic phase was removed and dried in a rotary evaporator. The dried residue was resuspended in organic solvent, and an ali-

were prelabeled with 0.4 pCi P ’ 4C-inositol .

CELLS WASHED 1. ADDITION OF EXTRACTION AND CELLS CULTURED IN DMEM + WrrH PBS * F ~ E ~ ~ o ~ ~ D ~ lDENTIFICAT,ON

10% FCS + myo- [U-14C]-INoSIToL PROWCTS 2. INCUBATION

I 1 1 I H H F 36HRS

Fig. 1. A schematic representation of the experimental protocol.

quot was counted to obtain the total radioactivity ex- tracted. The residue containing the 14C-poly-PtdIns from each time point was redissolved in 1 ml of ChCl,/CH30H (2:l,v/v). Separate aliquots were applied to activated thin layer chromatographic (TLC) plates coated with silica gel (0.25-mm thickness), which had previously been developed in the solvent system of CH30WH2O (40:60,v/v) containing 1.2 g potassium oxalate. The spotted plates were dried and developed in the solvent system of: chlo- roformJmethanolJlO% methylamine/O. 1 M ammonium acetate (40:28:5:2, v/v). Radioactivity on each plate was scanned and analyzed by the Berthold automatic TLC linear analyzer, model LB 2832, equipped with an Apple IIe computer.

Hydrolytic products containing 14C-inositol phosphates. The medium containing released 14C-inositol phosphates was collected and neutralized with 0.1 N NaOH. An aliquot was taken in aquasol to determine total radioactivity by counting in a Beckman liquid scintillation counter. The remaining 14C-medium was sub- jected to a modified ion exchange chromatographic system previously described by Berridge et al. (1983) for separating water-soluble 14C-inositol phosphates (InsP, InsP2, and InsP3). Briefly, the aqueous phase which contained the radioactive hydrolytic products was neutralized with 0.1 N NaOH to pH 7-8, and then applied to a column containing a 1-ml slurry of an ion exchange gel, AGl-x4 (formate form), 200-400 mesh, obtained from Bio-Rad Laboratories, Richmond, CA. The column was first eluted with 18 ml of H2O to remove free I4C-inositol. Subsequent fractions were eluted and collected with: 1) 15 ml of buffer containing 5 mM sodium tetraborate and 60 mM sodium formate (pH 9.0) which elutes the 14C-glycerophosphoinositol; 2) 9 ml of buffer containing 0.2 M sodium formate in 0.1 M formic acid which elutes 14C-inositol phosphate (InsP); 3) 9 ml of 0.4 M sodium formate in 0.1 M formic acid which elutes the 14C-inosi- to1 bisphosphate (InsPz); and 4) 9 ml of 1.0 M sodium formate in 0.1 M formic acid which elutes 14C-inositol trisphosphate (InsPs). A typical separation of these water-soluble hydrolytic products of polyPtdIns is shown in Figure 2. Aliquots were taken from each fraction and radioactivity was determined by counting in a liquid scintillation counter using aquasol, as described above.

Phosphorus assay Phosphorus assay of the cells was carried out accord-

ing to the method of Rouser et al. (1970). Sodium phosphate was used as standard.

RESULTS Incorporation of 14~-inositol into primary mouse

keratinocytes in culture My~-[U-~~C]-inositol was linearly incorporated into the

polyPtdIns of the proliferating cells during the first 36

la,25(OH)z VIT. D3 AND KERATINOCYTE F'tdIns 133

H 2 O

INOSITOL

yo 1 . 0 .

,- x \

0 . i Y 0 .1Y 0.1Y ncoow HCOOH/ HCOOH/

0 .06Y HCOONJ 0 .2Y 0.4Y 1.OY 0.005Y Na-T.1 H C 0 0 N a HCOON. HCOONa

b t- 3 2 4 0 i

FRACTION NUMBER

Fig. 2. Chromatographic separation of the soluble inositol phosphates on an ion exchange gel column.

hr after plating (Fig. 3), after which time the rate of incoporation of radiolabel into the polyPtdIns was ac- companied by an accumulation of the water-soluble 14C hydrolytic products (inositol phosphates) a t approxi- mately 36 hr. Concurrent morphological evaluations of the cells, as reported previously (data not shown) during the incubation period, revealed that immediately after 36 hr, the keratinocytes manifested evidence of differentiation; they were characterized by the appearance of cornified envelopes and thickened keratin bundles (Breitkrentz et al., 1981).

Dose-dependent effects of la,25(OH)~D3 on the generation of total 14C-inositol phosphates from

labeled keratinocytes These initial experiments were carried out to deter-

mine an appropriate concentration of la,25(OH)zD3 for use in subsequent experiments on the rapid stimulus effect of the vitamin D3 metabolite on hydrolysis and generation of individual inositol phosphates. Incubation of prelabeled cells with various amounts of la,25(OH)2& resulted in a concentration-dependent stimulatory effect on the generation of total 14C-inositol hosphates a t both the low concentration of 12 x 10 -' M as well as a t the maximal concentration of 12 x 10-l' M when compared to non-vitamin-incubated controls (Fig. 4). The release of inositol phosphates by 12 x 10 -lo M of la,25(OH)D3 is statistically significant (p < 0.05) when compared to non-vitamin-treated cells. At a high concentration of 12 x lo-' M, the vitamin metabolite de- creases release of the 14C total inositol phosphates towards the control level.

8

4 P R O L I F E R A T I V E + +- D I F F E R E N T I A T I N G v, 3 1. MODE I MODE a 2 0 n I " F I I T

P t d l n s

0 2 4 4'8

HOURS AFTER LABELING

Fig. 3. Time course of the incorporation of 14C-inositol into total InsPs and polyPtdIns of primary culture of mouse keratinocytes. Keratino- cytes (6 x 106/60-mm plate) in 3 ml of DMEM supplemented with 10% FCS were incubated at 37°C with 0.4 pCi of my~-[U-~~C]-inositol (53.3 mCiimo1) in each 60-mm petri dish with Liclz (10 mM) for the indicated successive time intervals. Cells from two plates were pooled for each sample. Radioactivity in both the aqueous and organic fractions was determined. Results were expressed as the percentage of 14C incorporated into labeled organic-solvent-soluble polyPtdIns and water-soluble InsPs. Each point represents the mean k S.D. of duplicate incubations from two separate experiments and are expressed as percentage over non-cell-containing control medium (at zero time). A , 0 radioactivity in organic lipid extracts; radioactivity in aqueous extract.

1

I 1

100 -1 2 -1 1 -10 - 9

Log-dose (M I

Fig. 4. Concentration-dependent effect of la,25(OH)2D3 -on the generation of total ''C-inositol phosphates from keratinocyte 14C-poly- PtdIns. Cells (6 x 106/60-mm plate) were incubated with 0.4 pCi of [U- 14]-inositol for 36 hr. Prelabeled cells with fresh medium were incubated with various concentrations of 1 a,25(OH)2D3 and Liclz (10 mM) at 37°C with 5% COz. Results are the mean k S.D. of duplicate experiments from three separate experiments. The data represent total ''C-inositol phosphates released from labeled cellular polyPtdIns and are expressed as percentage over control, which is taken as 100%. The generation of inositol phosphates at 12 x 10-l' M was statistically significant (p < 0.05) when compared to non-vitamin-treated cells.

134 TANG, ZIBOH, ISSEROFF, AND MARTINEZ

Short-term effects of la,25(OH)~D3 on the hydrolysis of keratinocyte 14C-PtdIns

In separate experiments, the time course of the effects of la %(OH)& at the maximal concentration of 12 x 10-ld on the hydrolysis of individual 14C-polyPtdIns was determined on keratinocytes previously labeled for 36 hr. The data in Figure 5 show a time course of the hydrolysis of individual inositol phospholipids of both the control and vitamin-treated cells. The effects of la,25(OH)& on the hydrolysis of PtdIns 4,5P2 and PtdIns 4P were rapid (maximal at 30 sec) and biphasic. At 30 sec, induced hydrolysis of PtdIns 4,5P2 (p < 0.05) and PtdIns 4P (p < 0.05) by la,25(OH)zD3 is statistically significant when compared to non-vitamin-treated cells. A notable feature of these results in that the control, unstimulated cells which contained no la,%(OH).$3 also revealed the hydrolysis of the PtdIns 4,5P2, presumably due to a small subset of keratinocytes which is already in the differentiation mode at 36 hr after plating.

Short-term effect of la,25(OH)& on the generation of individual 14C-inositol hosphates from keratinocytes B C-PtdIns

Simultaneous evaluation of 14C released into the incubation medium from the same experiments (as described above) revealed that la,25(OH) D3 increased hydrolysis of cellular 14C-PtdIns4,5P2 andq4C--PtdIns4P when compared to non-vitamin-treated cells. The hydrolysis of these two PtdIns was paralleled by the increased generation of 14C-InsP3 (maximal at 30 sec) and 14C-InsP2 (60 seconds), respectively, as illustrated in Figure 6. At 30 sec, the generation of InsP3 (p < 0.0005), InsP2 (p < 0.0025), and IP (p < 0.0025) by la,25(OH)& is statistically significant when compared to non-vitamin-treated cells. Interestingly, 1a,25(OH)zD3 exerted

minimal effect on the hydrolysis and generation of InsP from PtdIns (a precursor for the more phosphorylated PtdIns) during these experiments.

DISCUSSION Data from the present studies demonstrate that the in

vitro addition of low concentrations of la,25(OH)& to murine keratinocytes in culture did increase the hydrolysis of the cellular I4C-labeled polyPtdIns over control non-vitamin-treated cells. The induced hydrolysis resulted in a dose-independent generation of 14C-labeled inositol phosphates, particularly InsP3 and InsP2 (Fig. 6). These rapid biochemical events by la,%(OH)& were novel and preceded the onset of detectable morphological changes of differentiation in these cells. The unstimulated cells (Fig. 5) also revealed the hydrolysis of the PtdInsPz. Presumably, this hydrolytic activity of the unstimulated cells represents a basal hydrolytic state of a primary culture in a dynamic state. Although at 36 hr after plating the cell population is comprised of predominantly round basaloid proliferating cells, the possibility cannot be ruled out that a small subset of cells which is already entering the differential mode is also present. It is this subset of cells that presumably contributes to the hydrolytic activity seen in Figure 5 . This basal hydrolytic activity is consistent with a previous observation is our laboratory (data not shown) in which a correlation was established between the hydrolysis of PtdIns4,5P2 and the onset of differentiation in a primary culture of keratinocytes.

Interestingly, la,25(OH)zD3 has recently been reported to enhance the differentiation of murine keratinocytes in culture (Hosomi et al., 1983). The stimulatory effect of la,25(OH)2D3, which is evident a t near physio- logical concentrations of lo-'' M, implies that at least one of the in vitro functions of the vitamin D3 metabolite

HYDROLYSIS OF POLYPHOSPHOINOSITIDES

1 0 LI I- z 0 0 LL 0

mr Ptdlns4,5P2

- ' F N o -20 -30 ', Dg

-40

-10 :b4h \ f & = 1 . l

-20 ', -20 T- - - - --- -30

-40 -40 '' /

TIME OF HYDROLYSIS (SECONDS)

-70

Fig. 5. Short-term effect of l c~ ,25 (OH)~D~ on the hydrolysis of individual 14C-labeled polyPtdIns by muriue keratinocytes in culture, Kerat- inocytes (6 X 106/60-mm plate) were prelabeled with 0.4 pCi of I4C- inositol at 37°C for 36 hr. At 36 hr, the 14C-labeled medium was aspirated and replaced with fresh medium. A time course of the hydrolysis of the cellular polyPtdIns was initiated by reincubation with or without lor,25(OH)zD3 (12 X 10-l' M) dissolved in ethanol to a concentration of 0.1% of incubation mixture and Lids (10 mM). Rapid

extractions were made of the remaining organic-solvent-soluble phosphorylated inositides at 0, 30, 60, and 120 sec. Results are expressed as the percentage of control of the individual polyPtdIns. Zero time is taken as control. Each point represents the mean & S.D. of duplicate incubations from two separate experiments. Hydrolysis of ptdIns4,5P2 (p < 0.05) and ptdIns4P (p < 0.005) at 30 sec by l~i,25(OH)~D3 is statistically significant when compared to non-vitamin-treated cells.

la,25(OH)z VIT. D3 AND KERATINOCYTE PtdIns 135

INOSITOL PHOSPHATES GENERATION OF l o o r InsPg 1

-50 -30 t -50 -30 t T i m e o f H y d r o l y s i s ( S e

100 1n.P

Fig. 6. Short-term effect of la,25(OH)2D~ on the release of individual 14C-inositol phosphates by murine keratinocytes in culture. Keratino- cytes (6 X 106/60-mm plate) were relabeled with 0.4 pCi of '*C-inositol at 37°C for 36 hr. At 36 hr, the C-labeled medium was aspirated and replaced with fresh medium. A time course of the release of individual ''C-inositol phosphate from 14C-polyPtdIns was initiated by reincubation with or without la,25(OH)zD3 (12 X lo-'' M j dissolved in ethanol to a concentration of 0.1% of incubation mixture. Rapid extraction

were made of 14C from medium a t 0, 30, 60, and 120 sec. Results are expressed as the percentage over control of the individual inositol phosphates. Zero time is taken as control. Each point represents the mean f S.D. of duplicate incubations from two separate experiments. The generations of InsPs (p < 0.0005), InsP2 (p < 0.0025), and InsP (p < 0.0025) at 30 sec is statistically significant when compared to non- vitamin-treated cells.

B

may be to modulate polyPtdIns turnover and generate inositol triphosphate. The increased generation of InsP3 may function to mobilize intracellular Ca2+ in these cells, thereby providing the initial signal which alone or in concert with other modulators triggers keratinocyte differentiation. The role of intracellular Ca2+ in regu- lating keratinocyte differentiation remains unknown. Although, a modulatory effect of extracellular Ca2+ on the induction of keratinocyte differentiation in culture has already been established (Hennings et al., 1980), the mechanism of such an extracellular stimulus is unclear. Further studies are necessary to clarify whether the la,25(OH)?D3 -enhanced increase in InsP3 generation also precipitates the release of intracellular Ca2+ in the treated cells and whether or not this increase in intracellular Ca2+ also precedes the onset of differentiation of these cells. No attempts were made in these studies to compare the effects of this hormone-like la,25(OH)zD3 with that of its natural vitamin D3 (cholescalciferol) or its other vitamin D3 analogs, principally because no receptors for the other vitamin D3 metabolites or analogs have been demonstrated in these cells and because a previous study from this laboratory (Ziboh et al., 1982) failed to demonstrate any appreciable effect of the natural Vitamin D3 on membrane-associated epidermal phospholipase A.

Because receptors for lo1,25(OH)$3 and its modulatory action on murine keratinocyte differentiation have been described, our studies tested the hypothesis that the previously reported la,25(0H)2Da-induced differentiation of these cells was via the hydrolysis of the polpPtdIns and the release of the inositol phosphates. We recognize that, although these studies have demonstrated that la,25(OH)2D3 functions in vitro to enhance

the rapid hydrolysis of keratinocyte polyPtdIns and the generation of InsP3, which correlated with the induced onset of differentiation of epidermal cells in culture, the in vivo significance of the pro-differentiation effects of this vitamin D3 metabolite remains to be determined.

ACKNOWLEDGMENTS We thank Ms. Roberta Howering for her excellent

secretarial skill in the typing and preparation of this manuscript. This work was supported in part by re- search grants AM-30679 and AM-260009 from the Na- tional Institutes of Health of the United States Public Health Service. R.R.I. was the recipient of Clinical In- vestigator award AM-01195 from the National Insti- tutes of Health.

LITERATURE CITED Abe, E., Miyaura, C., Sakegani, H., Takeda, M., Konnon, K., Yamazaki

T., Yoshiki, S., and Suda, T. (1981) Differentiation of mouse myeloid leukemia cells induced by la,25-dihydroxy-vitamin Ds. Proc. Na- tional Acad. Sci. U.S.A 78:4990-4994.

Berridge, M.J., Dawson, R.M.C., Downes, C.P., Heslop, J.P., and Irvine, R.F. (1983) Changes in the levels of inositol phosphates after agonist- dependent hydrolysis of membrane phosphoinositides. Biochem. J., 212:473-482.

Breitkrentz, D., Boukamp, P., Leuder, M., and Fusening, N.E. (1981) Morphological and biochemical criteria for keratinization in primary and permanent mouse epidermal cell cultures. Front Matrix Biol., 9:57-82.

Clemens, T.L., Horiuchi, N., Nguyen, M., and Holick, M.F. (1981) Binding of Ia,25-dihydro~y-[~H]vitamin D3 in nuclear and cytosol fractions of whole mouse skin in vivo and in vitro. FEBS Lett., 134t203-206.

DeLuca. H.F.. and Schnoes, H.K. (1976) Metabolism and mechanism of action' of vitamin D. Annu. Rev. Biochem., 45t631-642.

Feldman, D., Chen, T., Hirst, M. Colston, K., Karasek, M., and Cone, C. (1980) Demonstration of la,25-dihydroxyvitamin D3 receptors in human skin biopsies J. Clin. Endocrinol. Metab., 51:1463-1465.

136 TANG, ZIBOH, ISSEROFF, AND MARTINEZ

Hennings, H., Michael, D., Cheng, C., Steinert, P., Holbrook, K., and Yuspa, S.H. (1980) Calcium regulation of growth and differentiaion of mouse epidermal cells in culture. Cell, 19:245-254.

Horiuchi, N., Clemens, T.L., Schiller, A.L., and Holick, M.F. (1985) Detection and developmental changes of the la,25(OH),D, receptor concentration in mouse skin and intestine. J. Invest. Dermatol., 84:461-464.

Hosomi, J., Hosoi, J., Abe, E, Suda, T., and Kuroki, T. (1983) Regulation of terminal differentiation of cultured mouse epidermal cells by lor,25- dihydroxyvitamin D3. Endocrinology 3:1950-1957.

Isseroff, R.R., Fusenig, N.E., and Rifiin, D.B. (1983) Plasminogen activator in differentiating mouse keratinocytes. J . Invest. Dermatol. 80:217-222.

MacLaughlin, J.A., Anderson, R.R., and Holick, M.F. (1982) Spectral character of sunlight modulates photosynthesis of previtamin D3 and its photoisomers in human skin. Science, 216:lOOl-1003.

Miyaura, C., Abe, E., Kurbayashi, T., Tanaka, H., Konnon, K., Nishii,

Y., and Suda, T. (1981) la,25-dihydroxyvitamin D, induces differentiation of human myeloid leukemia cells. Biochem. Biophys. Res. Commun., 102937-943.

Rouser, G., Fleischer, S., and Yamamoto, A. (1970) Two dimensional thin layer chromatographic separation of polar lipids and determi- nation of phospholipids by phosphorus analysis of spots. Lipids, 5494- 496.

Simpson, R.U., and DeLuca, H.F. (1980) Characterization of a receptor- like protein for 1-, 25-dihydroxyvitamin D3 in rat skin. Proc. Natl. Acad. Sci. U S A . 7758224826.

Yuspa, S.H., and Harris, C.C. (1974) Altered differentiation of mouse epidermal cells treated with retinyl acetate in vitro. Exp. Cell Res., 86:95-105.

Ziboh, V.A., Holick, J.A., McLaughling, C.L., Marcelo, C.L., and Voor- hees, J.J. (1982) Epidermal phospholipase Az: Activation by photo- lytic products of 7-dehydrocholesterol. J . Invest. Dermatol., 78:357.

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Novel regulatory actions of 1α,25-dihydroxyvitamin D3 on the metabolism of polyphosphoinositides in murine epidermal keratinocytes