1alpha ,25-Dihydroxyvitamin D3 Induces Sphingomyelin Hydrolysis in HaCaT Cells via Tumor Necrosis Factor alpha *

(Received for publication, October 3, 1996, and in revised form, January 23, 1997)

Christoph C. Geilen Dagger §, Meryem Bektas Dagger , Thomas Wieder Dagger par , Vitam Kodelja Dagger , Sergji Goerdt Dagger and Constantin E. Orfanos Dagger

From the Dagger  Department of Dermatology and the par  Institute of Molecular Biology and Biochemistry, University Medical Center Benjamin Franklin, The Free University of Berlin, D-12200 Berlin, Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Treatment of the human keratinocyte cell line HaCaT with 1alpha ,25-dihydroxyvitamin D3 (1,25-(OH)2D3) resulted in the hydrolysis of sphingomyelin with peak elevations of ceramide levels after 2-3 h (Geilen, C. C., Bektas, M., Wieder, Th., and Orfanos, C. E. (1996) FEBS Lett. 378, 88-92). In the present paper, the mechanism underlying the effect of 1,25-(OH)2D3 on sphingomyelin hydrolysis was investigated. Using the cell culture supernatant of HaCaT cells treated with 1,25-(OH)2D3 for 2 h, it was possible to induce sphingomyelin hydrolysis as early as 30-60 min after addition to resting cells. Several lines of experimental evidence indicated that tumor necrosis factor alpha  (TNFalpha ) mediates sphingomyelin hydrolysis after 1,25-(OH)2D3 treatment: (i) 1,25-(OH)2D3 stimulated TNFalpha mRNA expression after 1 h, (ii) newly synthesized TNFalpha occurred 2 h after 1,25-(OH)2D3 treatment, (iii) indirect activation of sphingomyelin hydrolysis by the supernatant of 1,25-(OH)2D3-treated HaCaT cells was abolished by preincubation of the supernatant with antibodies directed against TNFalpha , and (iv) preincubation of HaCaT cells with neutralizing antibodies directed against the 55-kDa receptor of TNFalpha blocked the ability of 1,25-(OH)2D3 to induce sphingomyelin hydrolysis in HaCaT cells. These data demonstrate that 1,25-(OH)2D3 activated sphingomyelin hydrolysis by an autocrine mechanism via TNFalpha expression. Furthermore, this indirect mode of action may serve as an explanation for the delayed induction of sphingomyelin hydrolysis by vitamin D3.


INTRODUCTION

1alpha ,25-Dihydroxyvitamin D3 (1,25-(OH)2D3)1 is the first compound identified as an inducer of reversible sphingomyelin (SM) hydrolysis in HL-60 human leukemia cells (1) leading to the generation of ceramide. Using cell-permeable ceramide analogues, it was possible to mimic the effects of 1,25-(OH)2D3 on cell proliferation and differentiation (2) and thereby confirm ceramide to be an important mediator of 1,25-(OH)2D3-induced HL-60 cell differentiation (3). In these studies, 1,25-(OH)2D3 caused SM hydrolysis in a time- and concentration-dependent manner, with a peak elevation in ceramide levels occurring 2 h after treatment of cells with 100 nM 1,25-(OH)2D3. After 4 h, both sphingolipids (SM and ceramide) returned to base-line levels by the transfer of a choline head group from phosphatidylcholine to ceramide (4). To date, several other agonists of the SM cycle have been described, including tumor necrosis factor alpha  (TNFalpha ), interferon-gamma , dexamethasone, interleukin-1, nerve growth factor, complement, and brefeldin A (5-8). In human keratinocytes and in the immortalized human keratinocyte cell line HaCaT, not only the well known inducers 1,25-(OH)2D3 and TNFalpha but also the vitamin D3 analogue calcipotriol activated SM hydrolysis. In this study, 1,25-(OH)2D3 and calcipotriol were shown to be late inducers of SM hydrolysis with peak levels after 3 h, whereas TNFalpha was shown to be an early inducer with peak levels after 30-60 min (9). However, the reason for these different time courses occurring in human keratinocytes as well as in HL-60 cells remained unclear, and the mechanisms underlying the coupling of these various extracellular inducers to activation of intracellular sphingomyelinase are largely unknown. In the case of TNFalpha , it was shown that interaction of TNFalpha with its 55-kDa receptor activates a phospholipase A2 generating arachidonic acid, which subsequently stimulates the cytosolic sphingomyelinase activity (10-12). As has been demonstrated recently, generation of ceramide then leads to activation of a protein kinase cascade, thereby implicating ceramide-activated protein kinase as a link between the TNF receptor and Raf1 (13). On the other hand, it is well established that 1,25-(OH)2D3 regulates gene expression via binding to an intracellular vitamin D receptor that belongs to the nuclear steroid hormone receptor superfamily (14, 15). Human keratinocytes are known to possess the vitamin D receptor (16), and 1,25-(OH)2D3 regulates the gene expression of several proteins in keratinocytes (17).

The aim of the present study was to investigate the mechanism responsible for the delayed effect of 1,25-(OH)2D3 on SM hydrolysis, and strong experimental evidence is provided that 1,25-(OH)2D3 induces SM breakdown indirectly via expression and secretion of TNFalpha in the human keratinocyte cell line HaCaT.


EXPERIMENTAL PROCEDURES

Materials

Streptomyces sp. sphingomyelinase, TNFalpha , phenylmethylsulfonyl fluoride, and leupeptin were purchased from Sigma (München, Germany). The TNFalpha ELISA kit was purchased from R & D Systems (Minneapolis), and anti-TNFalpha receptor (human) antibodies were from Bender MedSystems (Vienna, Austria). Solvents and reagents (reagent grade) were obtained from Merck (Darmstadt, Germany) and Fluka (Neu-Ulm, Germany), and Triton X-100 was purchased from Aldrich (Steinheim, Germany). [methyl-3H]Choline chloride (2.8-3.1 TBq/mmol), [alpha -32P]dCTP (110 TBq/mmol), and L-35S-labeled Pro-mixTM (in vitro cell labeling mixture) (>37 TBq/mmol) were from Amersham (Braunschweig, Germany). Anti-TNFalpha (human) antibodies were purchased from PeproTech Inc. (London, United Kingdom). 1,25-(OH)2D3 was a gift from Dr. Lise Binderup (Leo Pharmaceutical Products, Copenhagen, Denmark).

Cell Culture

HaCaT cells (18) were grown in Roswell Park Memorial Institute medium supplemented with 10% heat-inactivated fetal calf serum, 0.35 g/liter glutamine, 100,000 IU/liter penicillin, and 0.1 g/liter streptomycin in plastic culture dishes (Nunc, Wiesbaden, Germany). Media and culture reagents were obtained from Life Technologies, Inc. (Karlsruhe, Germany). Penicillin and streptomycin were from Boehringer (Mannheim, Germany). For experimental purposes, HaCaT cells were either maintained in keratinocyte basal medium (KBM) or in keratinocyte growth medium that was prepared from KBM by the addition of 10 ng/ml epidermal growth factor, 5 µg/ml insulin, 0.5 µM hydrocortisone, 50 µg/ml bovine pituitary extract, 100 µg/ml penicillin/streptomycin, and 2.5 µg/ml Fungizone. KBM and supplements were purchased from Clonetics Corp. (San Diego, CA). 1,25-(OH)2D3 and TNFalpha were diluted into KBM from 10 µM stock ethanol solutions or from 600 nM stock solutions containing 0.1% bovine serum albumin, respectively. NGF was diluted into KBM from 7.5 µM stock solution containing 0.1% bovine serum albumin.

Labeling of Cells

For choline labeling of HaCaT cells, medium was removed, and pulse medium (keratinocyte growth medium containing 3.7 × 104 Bq/ml [methyl-3H]choline) was added. After incubation for 72 h, cells were washed twice with phosphate-buffered saline (PBS) and then treated with 100 nM 1,25-(OH)2D3 in KBM. Cells were harvested in 400 µl of ice-cold PBS by the use of a cell lifter (Costar Corp., Cambridge, MA). After freeze-drying of the cells, lipids were extracted by a modified method of Bligh and Dyer (19) as described (20). Total lipid extracts were dried under a stream of nitrogen and stored at -20 °C.

Sphingomyelin Quantitation

SM was quantified using bacterial sphingomyelinase to release [3H]phosphocholine as described recently (12). Briefly, the total lipid extracts were resuspended in 100 µl of assay buffer (100 mM Tris-HCl, pH 7.4, 6 mM MgCl2, and 0.1% Triton X-100). The samples were sonicated for 5 min, and 1 unit/ml sphingomyelinase from Streptomyces sp. was added. After incubation at 37 °C for 2 h, the reaction was stopped by the addition of 1 ml of chloroform/methanol (2:1, v/v), and the liberated [3H]phosphocholine was recovered with the aqueous phase of a Folch extraction (22). Phase separation was completed by the addition of 100 µl of water, and the aqueous phase was taken for scintillation counting. The radioactivity of the aqueous phase normally reached 10,000 cpm, and the radioactivity in control samples was set as 100%. Subsequently, SM in the samples of 1,25-(OH)2D3-, TNFalpha -, and NGF-treated cells was calculated as percent of control. The assay conditions described above yielded maximal SM hydrolysis (>98%) without accompanying phosphatidylcholine hydrolysis (<5%) (data not shown).

Northern Blot

Confluent HaCaT cells were incubated with 100 nM 1alpha ,25-(OH)2D3 in KBM. After different incubation times, cellular mRNA was extracted from cells using a commercially available kit from Stratagene (Heidelberg, Germany) according to the instructions of the manufacturer, and then separated by electrophoresis in a 6% formaldehyde, 1.5% agarose gel and blotted onto a nylon membrane (GeneScreen Plus, DuPont). The filters were probed with the cDNA in question and reprobed using a beta -actin cDNA probe. The Northern blot was hybridized (23) with either of the following double-stranded cDNA probes: a 444-base pair polymerase chain reaction (PCR) DNA fragment covering nucleotides 404-847 of human TNFalpha or an 838-base pair PCR DNA fragment covering nucleotides 294-1131 of human beta -actin. Both probes were generated using PCR-Amplimers (Clontech, Palo Alto, CA) and a PCR amplification kit (Perkin-Elmer, Überlingen, Germany) (24). The cDNA probes were labeled with [alpha -32P]dCTP using the random priming kit RPN 1601 (Amersham-Buchler, Braunschweig, Germany) (25). Hybridizations were carried out overnight under stringent conditions at 60 °C in 1 M NaCl, 10% dextran sulfate, 1% SDS, 100 µg/ml salmon sperm DNA, and 2-4 × 105 cpm of the respective probes/ml (26). The filters were then washed twice at room temperature in 2 × SSC (0.3 M NaCl, 30 µM sodium citrate 2-hydrate, pH 7.0) plus 0.5% SDS followed by washing twice in the same solution at 60 °C for 30 min. Finally, the filters were washed twice in 0.1 × SSC for 5 min. Autoradiography was carried out at -80 °C using Kodak XAR/5 film with an intensifying screen.

TNFalpha ELISA

Confluent HaCaT cells were washed twice with PBS and treated with 100 nM 1,25-(OH)2D3 in KBM. After different incubation times, an aliquot of the cell culture medium was removed from cells by centrifugation. TNFalpha was detected in the supernatant by a commercially available ELISA kit (QuantikineTM, R & D Systems). 100 nM 1,25-(OH)2D3 in KBM was used as a blank. Detection of standard TNFalpha was linear over the range 2-25 pg/ml.

Indirect Stimulation of HaCaT Cells

Confluent HaCaT cells were stimulated with 100 nM 1,25-(OH)2D3 in KBM for 2 h. The cell culture medium was removed, clarified from cells by centrifugation, and added to confluent HaCaT cells that have been previously labeled for 72 h with 3.7 × 104 Bq/ml [3H]choline. Cell culture medium from HaCaT cells stimulated with 1% ethanol was used as control. After different time periods, cells were washed with ice-cold PBS and harvested. The SM content of the samples was then determined as already described above.

Immunoprecipitation of TNFalpha from the Supernatant of 1,25-(OH)2D3-treated Cells

For this, confluent HaCaT cells were stimulated with 100 nM 1,25-(OH)2D3 in KBM for 2 h. The cell culture medium was removed, briefly centrifuged, and treated with rabbit anti-human TNFalpha antibodies (PeproTech Inc.) or PBS as control at 4 °C for 2 h under moderate shaking conditions. The immune complex was precipitated with protein A-Sepharose for 1 h at 4 °C, and the protein A-Sepharose complex was sedimented by centrifugation. Control supernatant was likewise treated with protein A-Sepharose. The supernatants were incubated once more with the respective antibodies or with PBS as control, and the procedure was repeated. Subsequently, confluent HaCaT cells that had been previously labeled for 72 h with 3.7 × 104 Bq/ml [3H]choline were washed twice with PBS and incubated with the immune-precipitated supernatant. After 10, 30, and 60 min cells were washed with ice-cold PBS and harvested, and the SM content of the samples was determined as already described above.

Radiolabeling and Immunoprecipitation of TNFalpha

For this, confluent HaCaT cells were preincubated for 2 h with cysteine/methionine-free Dulbecco's minimal essential medium (Life Technologies, Inc.). The cells were labeled with 10 µCi/ml L-35S-labeled Pro-mixTM (in vitro cell labeling mixture) for 2 h and subsequently treated with 100 nM 1,25-(OH)2D3 or 1% EtOH as a control. After different times, cell culture supernatant was removed, and the cells were washed twice with PBS and harvested in 2 ml of 50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM phenylmethylsulfonyl fluoride, and 5 µg/ml leupeptin. 20 µl of anti-TNFalpha antibody (Sigma) were then added to 2 ml of cell culture supernatant of 2 ml of cellular extract, and the samples were incubated overnight at 4 °C. To immunoprecipitate TNFalpha , 5 mg of protein A-Sepharose were used (27). After 5 h at 4 °C under moderate shaking conditions, the protein A-Sepharose immune complex was sedimented by centrifugation at 4 °C and washed 3 times with ice-cold PBS. Finally, 90 µl of non-reducing SDS sample buffer were added, and the samples were boiled for 5 min. The protein A-Sepharose was sedimented by centrifugation, and the supernatant was loaded on SDS-polyacrylamide gel electrophoresis (15% acrylamide) and electrophoresed (28). The gels were dried, and the bands corresponding to TNFalpha were visualized and analyzed using a PhosphorImager (Molecular Dynamics GmbH, Krefeld, Germany). Under the condition described above, TNFalpha migrated with an apparent molecular mass of 17 kDa, which was confirmed with prestained SDS-polyacrylamide gel electrophoresis standards (Bio-Rad).

Blocking of the TNFalpha Receptor

Confluent HaCaT cells that had been previously labeled for 72 h with 3.7 × 104 Bq/ml [3H]choline were washed twice with PBS and then pretreated for 2 h with purified neutralizing antibodies against the human 55-kDa TNFalpha receptor, non-immune serum, or 0.1% bovine serum albumin in KBM as control. Subsequently, 1,25-(OH)2D3 in ethanol or ethanol vehicle alone was added to the cell culture medium to achieve a concentration of 100 nM and 1%, respectively. After 3 h of incubation, cells were washed with ice-cold PBS and harvested. SM was then quantified as described above.

Other Procedures

Statistical comparisons were made in these studies with Student's t test.


RESULTS AND DISCUSSION

Different Time Courses of Sphingomyelin Hydrolysis after TNFalpha and 1alpha ,25-Dihydroxyvitamin D3 Stimulation

Treatment of HaCaT cells with 100 nM 1,25-(OH)2D3 or the 1,25-(OH)2D3 analogue calcipotriol resulted in a time-dependent increase of sphingomyelin hydrolysis with a maximum after 3 h that was accompanied by an elevation of cellular ceramide levels (9). To establish the kinetics of the effect of 1,25-(OH)2D3 and TNFalpha on SM hydrolysis in the cells utilized for the following studies, an initial time course study was performed. Similar to previous studies, 1,25-(OH)2D3 induced a significant decrease of SM levels after 3 h of treatment, whereas in the case of TNFalpha , SM turnover of 12% was observed as early as 30 min following treatment of cells (Table I). Maximal effects of SM hydrolysis were observed 1 h after treatment with 30 nM TNFalpha . These different time courses of 1,25-(OH)2D3 and TNFalpha are in accordance with results obtained in the leukemic cell line HL-60 (1, 12, 29), which clearly demonstrated that 1,25-(OH)2D3 is a late and TNFalpha an early inducer of SM hydrolysis. It is interesting that the early induction of SM hydrolysis by TNFalpha is even more pronounced in the human acute leukemic Jurkat T cell line and the human monocytic cell line U937, which showed SM turnover with maximal effects after 2-3 min (30). In additional experiments, NGF was tested for its ability to induce SM hydrolysis. As shown in Table I, there was no effect of NGF on SM turnover of HaCaT cells after 0.5, 1, and 3 h, and the slight decrease of SM levels after 6 h was not significant.

Table I.

Time-dependent sphingomyelin hydrolysis after TNFalpha , 1alpha ,25-dihydroxyvitamin D3, and NGF stimulation

HaCaT cells were labeled with [3H]choline for 72 h, and then stimulated with 100 nM 1,25-(OH)2D3, 30 nM TNFalpha , or 15 nM NGF in KBM in a time-dependent manner. After incubation, cells were washed with PBS and harvested, and SM was measured as described under "Experimental Procedures." The data points represent the mean ± S.D. (n = 3) and are given as percent of control.


Time 1,25-(OH)2D3 TNFalpha NGF

h % control % control % control
0 100  ± 7 100  ± 10 100  ± 8
0.5 106  ± 11 88  ± 4 90  ± 5
1 103  ± 11 76  ± 5a 96  ± 5
3 74  ± 7b 104  ± 10 90  ± 8
6 104  ± 8 93  ± 12 83  ± 12

a Significantly different from controls at p < 0.05.
b Significantly different from controls at p < 0.02.

Indirect Stimulation of Sphingomyelin Hydrolysis by 1alpha ,25-Dihydroxyvitamin D3

Considering that 1,25-(OH)2D3 modulates gene transcription, it seemed reasonable that the late activation of SM hydrolysis by 1,25-(OH)2D3 occurs indirectly via expression and secretion of a mediator. To test this hypothesis, HaCaT cells were treated with 1,25-(OH)2D3 for 2 h, then the cell culture supernatant was removed and added to resting HaCaT cells. As shown in Fig. 1, the supernatant of 1,25-(OH)2D3-treated HaCaT cells induced SM hydrolysis as early as 20 min after addition to the cells with peak levels after 60 min. In an additional control experiment, HaCaT cells were treated with 1,25-(OH)2D3-containing medium that had been preincubated for 2 h in plastic culture dishes, and no significant decrease of SM was observed after 20-60 min of incubation (data not shown; see also Table I)


Fig. 1. Indirect stimulation of sphingomyelin hydrolysis by 1alpha ,25-dihydroxyvitamin D3. Confluent HaCaT cells were stimulated with 100 nM 1,25-(OH)2D3 in KBM for 2 h. The supernatant was removed and centrifuged. Resting HaCaT cells that had been previously labeled with [3H]choline were then washed twice with PBS and treated with the supernatant in a time-dependent manner. After incubation, the cells were washed with ice-cold PBS and harvested, and SM was measured as described under "Experimental Procedures." Values represent the mean ± S.D. (n = 3) and are each given as percent of control. *, significantly different from controls at p < 0.05; **, significantly different from controls at p < 0.01.
[View Larger Version of this Image (17K GIF file)]


Besides 1,25-(OH)2D3, additional activators of SM hydrolysis are described (e.g. TNFalpha , interleukin-1, interferon-gamma , NGF, or dexamethasone (6, 7)). In this context, it is known that interleukin-1 is down-regulated and NGF is up-regulated by 1,25-(OH)2D3 (17). Thus NGF was a possible candidate for the suggested mediator of indirect 1,25-(OH)2D3 action. As described above, however, NGF did not influence SM hydrolysis in HaCaT cells (Table I) indicating that this growth factor does not mediate 1,25-(OH)2D3-induced effects in SM hydrolysis. On the other hand, preliminary results showed that TNFalpha expression may also be increased by 1,25-(OH)2D3.2 To test if 1,25-(OH)2D3 indirectly stimulated SM hydrolysis via TNFalpha , confluent HaCaT cells were treated with 100 nM 1,25-(OH)2D3 for 2 h. The cell culture supernatant was then harvested, TNFalpha was immunoprecipitated, and the supernatant was investigated for its ability to induce SM hydrolysis in choline-prelabeled HaCaT cells. As shown in Table II, precipitation of TNFalpha abolished the ability of the supernatant of 1,25-(OH)2D3-treated HaCaT cells to induce SM hydrolysis after 30 and 60 min.

Table II.

Effect of immunoprecipitation of TNFalpha from the supernatant of 1alpha ,25-dihydroxyvitamin D3-stimulated HaCaT cells on sphingomyelin hydrolysis

Confluent HaCaT cells were stimulated with 100 nM 1,25-(OH)2D3 for 2 h. The cell culture medium was removed and treated with antibodies against TNFalpha or PBS as control. The immune complexes were precipitated with protein A-Sepharose. 1 × 106 [3H]choline-labeled HaCaT cells were then washed with PBS and incubated with the culture supernatants prepared as indicated above. After different incubation times, the cells were washed with ice-cold PBS and harvested, and SM was determined as described under "Experimental Procedures." Values represent the mean ± S.D. (n = 3) and are given as percent of control.


Time Treatment
 -Antibody +Antibody

min
0 100  ± 3.6 100  ± 3.6
10 94  ± 2.2 94  ± 6.6
30 84  ± 3.5a 95  ± 7.7
60 90  ± 4.1b 96  ± 16

a Significantly different from controls at p < 0.01
b Significantly different from controls at p < 0.05. 

1alpha ,25-Dihydroxyvitamin D3 Induces mRNA Expression and TNFalpha Secretion

From the data obtained, we focused our interest on TNFalpha and the time courses of TNFalpha mRNA expression. TNFalpha medium levels and TNFalpha biosynthesis were investigated in HaCaT cells after stimulation with 1,25-(OH)2D3.

As shown in Fig. 2, 1,25-(OH)2D3 induced TNFalpha mRNA expression after 1 h, and the signal diminished after 2-3 h. These findings were confirmed by amplification of the isolated mRNA using reverse transcription-PCR and detection of TNFalpha cDNA by Southern blot (data not shown). In these experiments, the slight increase of TNFalpha mRNA after 3 h of incubation that is seen in Fig. 2 was not observed.


Fig. 2. 1alpha ,25-Dihydroxyvitamin D3 induces TNFalpha mRNA expression. Confluent HaCaT cells were incubated with 100 nM 1,25-(OH)2D3. After different incubation times, the cellular mRNA was isolated, separated by a 1.5% agarose gel, and blotted onto a nylon membrane. The membrane was hybridized with a [alpha -32P]dCTP-labeled TNFalpha or beta -actin probe. Autoradiography was carried out for 1 week for TNFalpha and 1 h for beta -actin at -80 °C using Kodak XAR/5 film with an intensifying screen. The experiment was repeated, and similar results were obtained.
[View Larger Version of this Image (89K GIF file)]


To investigate TNFalpha biosynthesis in response to 1,25-(OH)2D3, HaCaT cells were labeled with [35S]cysteine/methionine. After different incubation times, TNFalpha was immunoprecipitated from the supernatant and cell lysates. Whereas in the cell lysates an elevation of labeled TNFalpha was detected after 1 h with a peak level after 2 h, labeled TNFalpha occurred in the medium after 2 h (Fig. 3). These data on biosynthesis were confirmed by determination of TNFalpha levels in the cell culture supernatant after treatment of HaCaT cells with 100 nM 1,25-(OH)2D3 using the ELISA technique as described under "Experimental Procedures," and an elevated level of 5.5 pg/ml TNFalpha occurred after 2 h. In ethanol-treated control cells, the level of TNFalpha was below the detection limit of 2 pg/ml.


Fig. 3. Determination of TNFalpha in the cell lysates and in the supernatants of [35S]cysteine/methionine-labeled HaCaT cells after treatment with 1,25-(OH)2D3. Confluent HaCaT cells were labeled with [35S]cysteine/methionine for 2 h, and 100 nM 1,25-(OH)2D3 was added. Control media contained 1% ethanol. After different time periods, cells (A) and medium (B) were harvested, TNFalpha was immunoprecipitated, and the immunoprecipitates were separated by SDS-polyacrylamide gel electrophoresis. Subsequently, the radioactivity was determined by a PhosphorImager. The experiment was repeated, and similar results were obtained.
[View Larger Version of this Image (28K GIF file)]


Neutralizing Antibodies against the 55-kDa TNFalpha Receptor Block 1alpha ,25-Dihydroxyvitamin D3-induced Sphingomyelin Hydrolysis

The data on TNFalpha production described above are in accordance with immunohistological studies that have shown that epidermal keratinocytes had intracellular plasma membrane and cytoplasmic labeling for TNFalpha (31). To investigate the role of TNFalpha in mediating the 1,25-(OH)2D3 effect on SM hydrolysis in more detail, the TNFalpha receptor was blocked by preincubation with neutralizing antibodies against the human 55-kDa TNFalpha receptor. This was done because it was shown in a previous study that the 55-kDa TNFalpha receptor is crucial for the effect of TNFalpha on SM hydrolysis (30). As shown in Table III, pretreatment of HaCaT cells with these neutralizing antibodies abolished 1,25-(OH)2D3-induced SM hydrolysis after 3 h, indicating that the mechanism underlying the stimulation of SM hydrolysis by 1,25-(OH)2D3 is mediated via TNFalpha . Preincubation of control cells with bovine serum albumin or non-immune serum did not influence SM hydrolysis. In this context, it is interesting to note that the 55-kDa but not the 75-kDa TNFalpha receptor was shown to be expressed in human keratinocytes (32). Human keratinocytes are therefore able to synthesize and simultaneously bind TNFalpha , thereby representing an autocrine loop. A similar production and auto-induction has been shown in keratinocytes for transforming growth factor alpha  (21).

Table III.

Effect of neutralizing antibodies against the 55-kDa TNFalpha receptor on 1alpha ,25-dihydroxyvitamin D3-induced sphingomyelin hydrolysis

Confluent HaCaT cells were labeled for 72 h with 3.7 × 104 Bq/ml [3H]choline. Cells were then washed twice with PBS and pretreated with KBM supplemented with purified neutralizing antibodies against the human 55-kDa TNFalpha receptor for 2 h. Control cells were pretreated with 0.1% bovine serum albumin in KBM. Subsequently, 100 nM 1,25-(OH)2D3 in KBM or ethanol vehicle in KBM as control was added to the cell culture medium. After 3 h of incubation, cells were washed with ice-cold PBS and harvested, and SM was quantified as described under "Experimental Procedures." Values represent the mean of six determinations ± S.D. and are given as percent of control.


Stimulation Pretreatment with TNFalpha receptor antibodies
 - +

Without 1,25-(OH)2D3 100  ± 5 100  ± 4
With 1,25-(OH)2D3 73  ± 12a 91  ± 11

a Significantly different from ethanol-treated controls at p < 0.01. 

In conclusion, the present study has demonstrated for the first time by several lines of experimental evidence that 1,25-(OH)2D3 indirectly induces the hydrolysis of SM. Furthermore, TNFalpha was shown to be the mediator of this indirect activation. The time course of 1,25-(OH)2D3-induced TNFalpha mRNA expression, TNFalpha biosynthesis, TNFalpha secretion, and TNFalpha -induced SM hydrolysis fit well with the time course of 1,25-(OH)2D3-induced SM hydrolysis.


FOOTNOTES

*   This work was supported in part by Grants SFB 366 and Ge641/3-3 from the Deutsche Forschungsgemeinschaft.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dedicated to Professor Werner Reutter on the occasion of his 60th birthday.


§   To whom correspondence should be addressed: Dept. of Dermatology, University Medical Center Benjamin Franklin, The Free University of Berlin, Hindenburgdamm 30, D-12200 Berlin, Germany. Tel.: 49-30-8445-2285; Fax: 49-30-8445-4262.
   Recipient of a grant from the Sonnenfeld-Stiftung.
1   The abbreviations used are: 1,25-(OH)2D3, 1alpha ,25-dihydroxyvitamin D3; KBM, keratinocyte basal medium; NGF, nerve growth factor; PBS, phosphate-buffered saline; SM, sphingomyelin; TNFalpha , tumor necrosis factor alpha ; PCR, polymerase chain reaction; ELISA, enzyme-linked immunosorbent assay.
2   C. Carlberg, personal communication.

ACKNOWLEDGEMENTS

We thank M. Hoffmann for excellent technical assistance and Dr. N. E. Fusenig, Deutsches Krebsforschungszentrum, Heidelberg, Germany, for the gift of HaCaT cells.


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