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The Clarke Institute of Psychiatry (C.W.Y.C., G.M.B.), Toronto, Ontario, M5T 1R8, Canada; Medical Research Council Membrane Biology Group (Y.S., M.S.), Department of Medicine, University of Toronto, Medical Science Building, Toronto, Ontario, M5S 1A8, Canada; Department of Medicine (M.W.), University of Toronto, Toronto, Ontario, M5S 1A8, Canada; and Department of Physiology (S.F.P.), University of Hong Kong, Hong Kong
Address all correspondence and requests for reprint to: Dr. M. Silverman, Medical Research Council Membrane Biology Group, Department of Medicine, University of Toronto, Medical Science Building, Toronto, Ontario M5S 1A8, Canada.
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Abstract |
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S (10 µM)
and pertussis toxin (100 ng/ml) provoked a marked decrease in binding
affinity (Kd was increased by a factor of 1.5–2.0), with
no significant difference in Bmax. Melatonin (1
µM) decreased the forskolin (10 µM)
stimulated cAMP level by 50%. HEK-293 cells do not express dopamine
D1A receptor. Following transient transfection of HEK-293 cells with
human dopamine D1A receptor (hD1A-R), exposure of the cells to dopamine
stimulated an increase in the level of cAMP. Similarly, transient
transfection of HEK-293 cells with rat glucagon-like peptide-1 (GLP-1),
glucose-dependent insulinotropic peptide (GIP), and PTH type 1
receptors, each resulted in an hormone inducible increase in cAMP
levels. Surprisingly, only the stimulatory effect of dopamine could be
inhibited by exposure to melatonin. The inhibitory effect of melatonin
on dopamine D1-induced increase in cAMP was completely inhibited by
pertussis toxin (100 ng/ml, 18 h). Immunoblot and
immunocytochemical studies were carried out using two polyclonal
antibodies raised against the extra and cytoplasmic domains of
Mel1a receptor. Immunoblot studies using antibody against
the cytoplasmic domain of Mel1a receptor confirmed the
presence of a peptide blockable 37 kDa band in HEK-293 cells. Indirect
immunofluorescent studies with both antibodies revealed staining
predominantly at the cell surface, but staining with the antibody
directed against the cytoplasmic domain required prior cell
permeabilization. By RT-PCR, HEK-293 cells express both
Mel1a and Mel1b messenger RNAs, but the
messenger RNA level for Mel1b is several orders of
magnitude lower than for Mel1a. We conclude that HEK-293 cells express MR predominantly of the Mel1a subtype. Our evidence suggests that one of the ways that melatonin exerts its biological function is through modulation of cellular dopaminergic responses.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To further explore the cellular and molecular mechanisms by which melatonin exerts its epithelial effects in greater detail, it would be advantageous to have an appropriate in vitro experimental system. Accordingly, several different kidney epithelial cell lines were screened for the presence of functional MR. Human embryonic kidney-293 (HEK-293) cells exhibited by far the highest 125I-MEL binding of the cells tested with level comparable to that reported in human kidney cortex (10) and were therefore chosen for further investigations.
HEK-293 cells were found to express an epithelial phenotype when compared with smooth muscle like mesangial cells as determined by intermediate filament characterization. The results of the present study show that HEK-293 cells express functional MR coupled to Gi predominantly of the Mel1a subtype and to a lesser extent of Mel1b as revealed by RT-PCR, pharmacological 125I-MEL binding studies and immunoblot studies with peptide specific polyclonal anti-Mel1a receptor antibody. When the effects of melatonin on stimulated cAMP were studied, melatonin decreased forskolin and dopamine stimulated cAMP via a pertussis toxin-sensitive mechanism. Studies carried out in HEK-293 cells transiently transfected with dopamine D1A, GIP, GLP-1, and PTH type 1 receptors, which coupled to adenylyl cyclase through stimulatory G protein (Gs), revealed that exposure to melatonin only affected the cAMP elevation elicited by dopamine. These results suggest that one of the mechanisms by which melatonin exerts its biological function is through modulation of cellular dopaminergic responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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S),
pertussis toxin, DTT, phenylmethylsulfonyl fluoride (PMSF), and other
chemicals of the highest chemical grade were obtained from Sigma
Chemicals (St. Louis, MO).
Cell lines
MDCK, LLC-PK1, OK and HEK-293 were from American
Type Culture Collection (ATCC) (Rockville, MD). They were grown in 100
mm culture dishes in DMEM supplemented with 10% FBS, 50 U/ml
penicillin and 50 µg/ml streptomycin in a humidified incubator with
5% CO2 at 37 C. The medium was changed two or three times
per week, and the cells were split 1:3 or 1:4 upon reaching confluence.
The culture conditions before each of the assays varied depending on
the experimental protocols and are described in the relevant sections.
Mesangial cells as mesenchymal, smooth-muscle like cells in the renal
glumerulus were prepared and cultured as previously described (13).
HEK-293 cells between passage 35 to 55 were used in the study.
DNA
The reporter vector, which codes for the cDNA of green
fluorescence protein (GFP), was obtained from Clontech (Palo Alto, CA).
The genomic DNA encoding the full length of hD1A-R subcloned into pCD
(6.5 kb) was a generous gift from Dr. H. B. Niznik of the
Department of Pharmacology, University of Toronto (Toronto, Ontario,
Canada). The full length rat PTH type 1 receptor (rPTH-R) cDNA
subcloned into mammalian expression vector pcDNA3 (5.4 kb; Invitrogen)
was donated by Dr. S. Palcy and Dr. D. Golzman at Calcium Research
Laboratory, Royal Victoria Hospital (Montreal, Quebec, Canada). The
full length of rat GLP-1 receptor (rGLP-1R) (14) and rat GIP receptor
(rGIP-R) (15) were also subcloned into the vector pcDNA3. The cDNAs of
Mel1a and Mel1b receptors subcloned into the
vector pcDNA1 and pcDNA3, respectively, are kindly donated by Dr.
S. M. Reppert at Laboratory of Developmental Chronobiology,
Massachusetts General Hospital (Boston, MA).
2-[125I]Iodomelatonin binding assays
The assays were performed in duplicate. Cells were washed with
HBSS three times and scraped and the suspension was centrifuged at
8,000 x g for 15 min. Cell pellets were washed twice
in 50 mM Tris-HCl buffer (pH 7.4), centrifuged at
12,000 x g for 10 min and were then resuspended in
Tris-HCl buffer for binding assays. Preliminary studies in our
laboratory suggested that crude membrane preparation from HEK-293 cells
yielded the same binding capacity as the cell suspension. Hence, for
convenience, we employed the later method in the present study.
Cell suspensions were incubated with 5–160 pM of
125I-MEL at 37 C for 1 h with or without melatonin (1
µM) in the saturation studies. For the kinetic studies,
incubation time varied from 5–100 min at a fixed concentration of
125I-MEL (50 pM). Melatonin (1
µM) is added at 40 min to initiate dissociation. For the
competition studies, indoles of various concentrations ranging from 1
pM to 10 µM were incubated with samples and
fixed concentration of 125I-MEL (70 pM). The
effects of GTPS (10 µM) and pertussis toxin (100
ng/ml, 18 h) on the saturation curves were also investigated. The
effect of pertussis toxin was performed by pretreating the cells with
100 ng/ml pertussis toxin at 37 C overnight. On the next day, the cells
were harvested as previously described. Cells pretreated with working
solution of the pertussis toxin were used as control.
All reactions were terminated by addition of 3 ml cold Tris-HCl buffer (pH 7.4) three times followed by immediate vacuum filtration through Whatman GF/B glass fiber filters (pore size 1.0 µm). Radioactivity was measured by a gamma counter (Auto-gamma Gamma Counting System, Packard Instrument Company, Meriden, CT) with an efficiency of 74%. The equilibrium dissociation constant (Kd), maximum number of binding sites (Bmax), dissociation rate constant (K-1), association rate constant (K1), and inhibition constant (Ki) were determined as reported (7, 16).
RT-PCR
RT-PCR was performed as previously described (13). Primers
specific for Mel1a and Mel1b receptors, keratin
no. 8, desmin, vimentin, and glyceraldehyde 3-phosphate dehydrogenase
(G3PDH) were selected for the RT-PCR (Table 1
). Primers for Mel1a and
Mel1b receptors were designed to contain sequences in the
transmembrane domain 1 and 2 of the receptors (17). Amplification of
genomic DNA would be unlikely to occur due to the presence of a large
intron between the transmembrane domains. Following RNA isolation from
HEK-293 and mesangial cells by RNeasy total RNA isolation kit, RNAs
were treated with DNase I to remove possible tracing amount of genomic
DNA. RT was then performed producing first strand cDNA. PCR was then
performed, and the products were subjected to 1% agarose gel
electrophoresis. Master mix with all the reagents except templates were
used in RT and PCR to ensure same reaction conditions for all
samples.
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Immunoblot study
HEK-293 cells were scraped in the cell lysate buffer (20
mM Tris-HCl, pH 8.0 at 4 C, 10% glycerol, 1% Triton
X-100, 137 mM NaCl) with proteinase inhibitors (1
mM EDTA, 1 mM PMSF, 1 mg/liter each of
leupeptin, pepstatin A, and aprotinin) and rocked at 4 C for 1 h.
Then the suspension was centrifuged at 20,000 x g for
20 min and the supernatant was collected for gel electrophoresis. Cell
lysates (80 µg/lane) were subjected to 10% SDS-PAGE in sample buffer
(62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 42
mM DTT, and 0.01% bromophenol blue) and transferred
electrophoretically to nitrocellulose. The nitrocellulose sheets were
blocked in TBS-T (20 mM Tris-HCl, 137 mM NaCl,
0.2% Tween-20, pH 7.6) with 5% nonfat dry milk for 30 min at room
temperature. Nitrocellulose strips were then incubated with
anti-Mel1a IgG-TIL3 (0.4 mg/liter) supplemented with or
without 0.5 mg/liter of the corresponding peptide TIL3 in blocking
buffer at room temperature for 1 h then washed with TBS-T at least
four times for a total of 1.5 h. The strips were then incubated
with a horseradish peroxidase conjugated goat antirabbit IgG (Bio-Rad,
Hercules, CA) followed by washing in TBS-T and detected by ECL
according to the manufacturer’s instruction. The immunoblot presented
in this paper is representative of three separate experiments.
Immunolocalization of MR using anti-Mel1a receptor
antibodies
HEK-293 cells were plated on coverslips and cultured for 1 day.
Cells were first rinsed in HBSS, then fixed in freshly prepared 3%
paraformaldehyde in PBS (pH 7.4) and blocked in PBS with 10% dry milk
followed by permeabilization with 0.2% Triton X-100 in PBS. Antibody
directed against Mel1a receptor (anti-Mel1a
IgG-TIL3 or anti-Mel1a IgG-TEL3) at concentration of 0.05
mg/ml was added and incubated for 30 min at room temperature. The IgG
fraction of preimmune sera was used as a control. PBS with 0.2%
Tween-20 and 1% Triton X-100 was used to wash the cells after
incubation, which were then incubated at dark with rhodamine-labeled
goat antirabbit IgG (Jackson ImmunoResearch Lab, Inc., West Grove, PA)
for 30 min at room temperature. Washing was repeated as described
previously. Cells were then mounted on glass slides with 0.1%
p-phenylenediamine with 90% glycerol and observed under a confocal
laser-scanning microscope (LSM-410, Carl Zeiss Jena GmbH, Oberkochen,
Germany) with a Carl Zeiss LSM 3.8 program.
Calcium phosphate transfection
The DNA for transfection was purified using the Qiagen Plasmid
Kit (Qiagen, Chatsworth, CA). HEK-293 cells were transfected with
different DNA separately, including the plasmid pCD encoding the
genomic DNA of hD1A-R, the plasmids pcDNA3 encoding the cDNA of
rGLP-1R, rGIP-R, and rPTH-R. Cells were plated at a density of 8
x 104 cells/35cm2 in the 6 well plates in DMEM
with 10% FBS one day before tranfection. DNA (1.8 µg/well) was
introduced into the cells as a calcium phosphate DNA complex using the
calcium-phosphate transfection kit (18). Empty plasmid, pCD or pcDNA3,
was used as a negative control in each experiment. The vector encoding
cDNA of GFP from the jellyfish Aequorea victoria was used as
a reporter for estimating gene expression efficiency (19) by counting
the number of GFP expressed per million of cells under fluorescence
microscope 72 h after transfection.
Adenylyl cyclase activity
Cells were grown to confluence in six-well plates in DMEM
with 10% FBS. Reactions were started by the addition of 10
µM forskolin and/or melatonin analogs at various
concentrations from 1 µM to 1 pM at 37 C for
30 min supplemented with 100 µM isobutylmethyl xanthine
(Sigma, St. Louis, MO). The reactions were terminated by centrifugation
of the cell suspension followed by the addition of 5 mM
acetic acid to the pellets. Then, cells were boiled for 5 min,
sonicated, and centrifuged for 15 min. The supernatant was collected
and assayed for cAMP, whereas the pellet was saved for protein assay
based on the method reported by Lowry et al. (20) with BSA
as the standard. All determinants were done in triplicate. Cyclic AMP
levels were determined in duplicate by RIA. The data are expressed as
the increment of cAMP above basal levels.
For the cells transfected with genomic DNA of the hD1A-R, cDNA of rGIP-R, rGLP-1R, or rPTH-R, stimulation was performed 72 h after transfection with the same protocol mentioned above. Cells were incubated with different concentrations of dopamine, shGIP, shGLP-1(7–36), and shPTH to induce cAMP accumulation and the EC50 values were determined. Those hormones were also added to the untransfected cells to check if the corresponding receptors are expressed endogenously and the effects of melatonin were also examined. Meanwhile, the effects of melatonin on transfected cells were studied by incubating cells with dopamine (10 µM), shGIP (10 nM), shGLP-1(7–36) (10 nM) or shPTH (10 nM) respectively in concentrations closed to their EC50 with or without melatonin (1 µM).
In some experiments, cells were pretreated with 100 ng/ml of pertussis toxin in DMEM. In the case for untransfected cells, incubation started on the day before stimulation whereas for the transfected cells, incubation started on the third day after transfection. After 18 h of incubation, the cells were washed twice with DMEM and stimulation was done as described earlier.
RIA of cAMP
The RIA was performed using a high specific activity adenosine
3',5'-cyclic phosphoric acid
2'-O-succinyl-3-[125I]iodotyrosine methyl
ester ([125I]cAMP-TME) together with a high affinity
antisuccinyl cAMP sera as reported (9, 21). All assays were performed
in duplicate.
Statistical analysis
Comparisons of two groups were analyzed by paired or unpaired
Student’s t test. Group differences in the cAMP studies
were analyzed by one-way ANOVA, followed by Fisher’s
least-significant-difference (LSD) tests. All analysis were performed
by Systat 5.2.1. (Systat Inc., Evanston, IL), fitted for the Macintosh
system. The level of significance were set at P <
0.05. Data are expressed as means ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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,
HEK-293 cells but not mesangial cells expressed keratin no. 8
consistent with cells being of epithelial origin. Both cell types
expressed vimentin consistent with its production by transformed cell
lines and mesenchymal cells (Table 1) (22). Mesangial cells also
expressed desmin consistent with its production by smooth muscle-like
cells (22). Contamination of genomic DNA was unlikely to occur in these
experiments because no amplification product was observed when PCR was
performed using G3PDH primers without reverse transcription
following RNA isolation (data not shown).
In vitro ligand-receptor binding studies of HEK-293 cells
The saturability of 125I-MEL binding was determined
using a range of 125I-MEL concentrations (5–160
pM). Binding of 125I-MEL to MDCK, OK, and
LLC-PK1 was too low to be detected (data not shown).
Specific binding of 125I-MEL to HEK-293 cells increased
with increasing concentration of radioligand and approached saturation
approximately at 80 pM 125I-MEL (Fig. 1a). A representative Scatchard plot is
showed in Fig. 1b. The Kd was 23.8 ± 0.5
pM, and the Bmax was 1.17 ± 0.11 fmol/mg
protein (n = 3). Hill coefficients approached 1.0 in each case
(Fig. 1c). Taken together, these results suggest the presence of a
single class of high affinity binding sites in HEK-293 cells.
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).
The order of potency is shown in order in the table.
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Specific binding of 125I-MEL to the membrane was inhibited
by the addition of 10 µM nonhydrolyzable GTP analog,
GTPS. The addition of GTPS significantly increased the
Kd from 23.8 ± 0.5 to 37.2 ± 0.9 pM
(P < 0.05, paired Student’s t test, n
= 3) and with no significant difference in the Bmax
(1.17 ± 0.11 and 0.91 ± 0.04 fmol/mg protein). Incubation
of HEK-293 cells with pertussis toxin (100 ng/ml) significantly
increased the Kd from 14.3 ± 3.3 to 31.7 ± 2.3
pM (P < 0.05, unpaired Student’s
t test, n = 3), whereas there was no significant
difference in the Bmax (0.40 ± 0.05 and 0.45 ±
0.07 fmol/mg protein).
Characterization of MR in HEK-293 by RT-PCR
As shown in Fig. 2 by PCR, HEK-293
cells can be seen to express messenger RNAs for both Mel1a
and Mel1b receptors.
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shows an immunoblot using
anti-Mel1a IgG-TIL3 antibody. A single peptide blockable
band at 37 kDa is present. The molecular mass of approximately 37 kDa
is consistent with that found in the human brain and guinea pig kidney
proximal tubule basolateral membrane (8) and close to the predicted
size of the MEL1a receptor from the cDNA clone (39.4 kDa)
(23). These observations together with the binding studies and RT-PCR
strongly suggest that HEK-293 cells express MR with majority of
MEL1a subtype.
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, a and b, using anti-Mel1a
IgG-TIL3 antiserum (antibody directed against a predicted intracellular
domain of the Mel1a receptor), specific staining was
observed only in permeabilized but not nonpermeabilized cells, and the
staining is localized primarily at the cell membrane.
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, c and d, application of
anti-Mel1a IgG-TEL3 antiserum (the antibody directed
against a predicted extracellular domain of the Mel1a
receptor) allowed staining of both the permeabilized and
nonpermeabilized cells.
Mechanism of melatonin action on HEK-293
In kidney (9), pars tuberalis (24), and other neural tissues (21, 25), it has been demonstrated that melatonin reduces forskolin
stimulated cAMP. Therefore, it is possible that melatonin might
exert its regulatory role on epithelia by regulating intracellular cAMP
levels. As shown in Fig. 5a, stimulation
of HEK-293 cells with 10 µM forskolin in the absence of
melatonin induced an approximately 10-fold increase in the cAMP level
(P < 0.05, unpaired Student’s t test,
n = 3). Exposure of HEK-293 cells to melatonin had no effect on
basal cAMP levels (data not shown) but caused a 50% reduction in
forskolin (10 µM) stimulated cAMP at a concentration of 1
µM (Fig. 5a). Furthermore, the melatonin-induced
reduction of forskolin stimulated cAMP was abolished by pretreatment
with pertussis toxin (Fig. 5a), implying that the effect mediated by
the MR transduction is Gi coupled.
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Application of 10 pM melatonin drastically decreased the
dopamine (10 µM) stimulated cAMP in the D1A receptor
transfected HEK-293 cells by 61% and further inhibition was observed
at 1 µM melatonin (Fig. 5b) (P < 0.01,
ANOVA, n = 3).
Similar to what was observed after exposure to forskolin, pretreatment
of transfected cultures with pertussis toxin for 18 h (100 ng/ml)
had no effect on dopamine-stimulated cAMP accumulation but
significantly reduced the inhibitory effect of melatonin (Fig. 5b).
As shown in Fig. 5b, all rGIP-R, rGLP-1R and rPTH-R transfected HEK-293
cells showed stimulated cAMP above basal level by corresponding
hormones (P < 0.01, unpaired Student’s t
test, n = 3), reaching levels approximately 50–80% of that
observed by dopamine. But interestingly, in contrast to the case for
dopamine, the increase in cAMP stimulated by shGIP, shGLP-1(7–36), and
shPTH was not inhibited by melatonin (Fig. 5b) (P >
0.05, unpaired Student’s t test, n = 3).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The molecular mechanisms of melatonin action on HEK-293 cells
were assessed by examining the effects of melatonin on cellular cAMP
levels. Pertussis toxin-sensitive inhibitory effects of melatonin were
observed only on forskolin and dopamine-stimulated cAMP accumulation,
suggesting that melatonin interaction with HEK-293 MR is mediated by a
Gi-coupled interaction. The effect of melatonin on forskolin stimulated
cAMP is in line with those reported in chicken kidney (9) and pars
tuberalis (24). However, what is particularly intriguing is that the
interaction of melatonin and dopamine exhibited a degree of specificity
in that melatonin could not inhibit the GIP-, GLP-1-, and
PTH-stimulated cAMP on HEK-293 cells. In other words, of the four
Gs-coupled hormone receptors tested, only dopamine exhibited an
interaction with melatonin. It is not so surprising that picomolar
concentrations of melatonin acting through endogenous MR are capable of
effectively blocking (
50% inhibition) of dopamine stimulated
HEK-293 overexpressing dopaminergic receptors because similar melatonin
concentrations yield approximately the same degree of inhibition
following forskolin stimulated cAMP. Nevertheless, it suggested the MR
signaling is more effective than the dopamine D1A receptor signaling
because a lesser level of MR was capable of competing with and blocking
the presumably massive stimulation from the overexpressed D1A
receptors. This may also explain the levels of MR in the kidney
in vivo are likely to be lower because a relatively low
level of MR would be enough to generate an effective signaling
response.
Although one study has suggested that MR upon stimulation by melatonin might directly inhibit adenylyl cyclase (28) and thereby decrease cAMP levels, it is more likely that in HEK-293 cells, endogenous MR and transfected dopamine D1A receptors interact with a common isozyme of adenylyl cyclase whereas GIP, GLP-1 and PTH receptors are coupled to different HEK-293 adenylyl cyclase isozymes. However, we have to emphasis that these experiments were done under artificial situation. It is of course possible that this specificity of the melatonin dopamine interaction is restricted to HEK-293 cells or in vitro situation only. But a likely scenario is that this observation has general biological implication. For example, one could hypothesize that the mechanism by which melatonin affects epithelial (as well as nonepithelial) functions is through modulation of their dopaminergic response. In fact, the melatonin-dopamine interaction has been described in other cells (5), but to our knowledge, the present study is the first demonstration of differential inhibitory effects of melatonin on dopamine-stimulated adenylyl cyclase.
It should also be pointed out that the melatonin-dopamine interaction might not be necessarily restricted to the level of cellular cAMP. Other mechanisms may also be involved. We believed that such specific melatonin-dopamine but not melatonin-GIP, GLP-1 or PTH interaction may suggest possible physiological functions of melatonin and also the mechanisms, besides adenylyl cyclase, involved in the melatonin-dopamine systems. Unfortunately, we could not distinguish which MR subtype was responsible for the effects in the study. However, we believe the Mel1a is also the predominant subtype of MR at the protein level based on the results of RT-PCR and was responsible for the effects. Nonetheless, future experiments have to be done to support our hypothesis.
Based on the present study, we would predict that the Gi-coupled Mel1a (and/or possibly also Mel1b) receptor and the Gs-coupled dopamine receptor share the same effector adenylyl cyclase isozyme. It may be that other hormone receptors also share this same isozyme, but the specificity of the linkage of melatonin and dopamine is sufficiently interesting to warrant further investigation to explore this hypothesis. The HEK-293 cell model should facilitate the dissection of the complex cellular and molecular processes of melatonin action on epithelia in the future.
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Footnotes |
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2 Recipient of a postdoctoral fellowship award from the Kidney
Foundation of Canada.
3 Ontario Mental Health Foundation Research Associate.
4 Member of the MRC Membrane Biology Group, Department of Medicine,
University of Toronto.
Received March 26, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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