1 Institute of Molecular Pharmacology, D-13125 Berlin, Germany
2 ASCA Angewandte Synthesechemie Adlershof GmbH, 12489 Berlin, Germany
3 Department of Pathology, University of Tennessee Health Science Center,
Memphis, TN 38163, USA
* Author for correspondence (e-mail: aslominski{at}utmem.edu)
Accepted 2 December 2002
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Summary |
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Key words: CRF, Urocortin, Keratinocytes, Melanocytes, Calcium signaling, Calcium oscillation
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Introduction |
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It is currently accepted that signal transduction through CRF-R is
primarily linked to the activation of adenylate cyclase followed by the
production of cyclic adenosine monophosphate (cAMP)
(Perrin and Vale, 1999; Speiss
et al., 1998). However, phospholipase C may also be activated, producing
inositol triphosphate (IP3), which in turn activates PKC-dependent
and calcium-activated pathways (Chalmers et
al., 1996
; Speiss et al., 1998). CRF, URC and CRF-Rs are widely
expressed in peripheral tissues, where CRF function may be important in the
local regulation of homeostasis (Linton et
al., 2001
; Slominski et al.,
2000d
; Slominski et al.,
2001
). The intracellular signaling pathway activated through
peripheral CRF receptors involves the production of cAMP and subsequent
activation of protein kinase A (PKA)
(Linton et al., 2001
;
Slominski et al., 2001
). The
involvement of calcium-activated pathways via phospholipase C or
membrane-bound calcium channels has also been demonstrated, and CRF
receptor-mediated activation of mitogen activated protein (MAP) kinase signal
transduction pathways has been reported in several cell types
(Linton et al., 2001
;
Slominski et al., 2001
).
Mammalian skin is both a source of and a target for CRF-related peptides
(Slominski et al., 2000c;
Slominski et al., 2001
), which
led to the proposal that the CRF/URC signaling system may play a central role
in the skin's response to stress
(Slominski et al., 1999
;
Slominski et al., 2000d
;
Slominski et al., 2001
); skin
produces CRF and URC and expresses CRF-R1
(Pisarchik and Slominski,
2001
; Roloff et al.,
1998
; Slominski et al.,
1995
; Slominski et al.,
1996
; Slominski et al.,
1998
; Slominski et al.,
2000a
; Slominski et al.,
2000c
). These CRF-Rs are functional, e.g., they respond to CRF and
URC through activation of receptor(s)-mediated pathways to modify skin cell
phenotype (Fazal et al., 1998
;
Quevedo et al., 2001
;
Slominski et al., 1999
;
Slominski et al., 2000b
;
Slominski et al., 2001
). Human
keratinocytes, both normal immortalized (HaCaT) and neonatal express CRF-R1
and bind CRF (Quevedo et al.,
2001
; Slominski et al.,
1999
; Slominski et al.,
2000b
; Slominski et al.,
2001
). Furthermore, CRF stimulated cAMP production and inhibited
the proliferation of HaCaT keratinocytes with a higher potency than either
urocortin or sauvagine (Slominski et al.,
2000b
). Similarly, CRF modulated the interferon induced expression
of hCAM and ICAM-1 adhesion molecules and of the HLA-DR antigen in neonatal
keratinocytes (Quevedo et al.,
2001
). In melanoma cells, CRF binding sites were detected, and CRF
and URC, added to cell suspensions, produced dose-dependent increases in
intracellular calcium. The effect showed a fairly rapid onset (within a
second) (Fazal et al., 1998
;
Slominski et al., 1999
); the
Ca2+ signal did not, however, return to the basal level, but rather
continued to increase gradually with time. To understand this phenomenon
better we investigated in detail the mechanism of CRF- and URC-mediated
calcium signaling in skin cells in situ, using laser confocal microscopy. This
technique has a high space resolution, which we exploited to investigate the
Ca2+ distribution in single peptide-stimulated cells.
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Materials and Methods |
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Cells
Semi-confluent HaCaT immortalized human keratinocytes were maintained at
37°C and 5% CO2 in DMEM medium supplemented with antibiotics in
the presence of 10% FBS as described previously
(Slominski et al., 2000b).
Human melanoma cells (SKMEL188) were grown in Ham's F10 medium following
standard methods; the media were supplemented with 10% fetal bovine serum and
antibiotics (Slominski et al.,
1998
). Normal human neonatal keratinocytes were a gift from
Mitchell Denning, Loyola Medical Center
(Quevedo et al., 2001
).
Primary cell cultures were established from foreskin as described previously
(Quevedo et al., 2001
). The
cells were propagated in low-calcium (0.15 mM), serum-free KGM containing BPE
(Quevedo et al., 2001
). Normal
human neonatal melanocytes (a gift of Z. Abdel-Malek, University of
Cincinnati, OH) were cultured in MCDB 153 supplemented with 4% FBS, 13
µg/ml BPE, 8 nM TPA, 1 µg/ml
-tocopherol, 0.6 ng/ml basic
fibroblast growth factor, 1 µg/ml transferrin, 5 µg/ml insulin and 1%
antibiotic-antimycotic mixture as described previously
(Abdel-Malek et al., 1995
).
For optical measurements, the cells were cultivated for 2 days on glass cover slips (30 mm diameter). After washing with phosphate buffered seline (PBS), cells were loaded with the Ca2+ indicator Fluo-3/AM (4.4x10-6 M in the culture medium (see above) in the presence of 0.01% Pluronic F-127) for 30 minutes at 37°C in darkness, then washed three times and incubated in the medium (see below for the measurements).
Optical Ca2+ measurements using confocal laser scanning
microscope
The present studies were performed utilizing an LSM 410 and an LSM 510
invert confocal laser scanning microscope (Carl Zeiss Jena, Germany) with a
x100/1.3 oil immersion objective and an argon-krypton laser (488 nm)
excitation source. The excitation wavelength was selected by a dichroitic
mirror (FT510). The fluorescence intensities were detected at wavelengths
greater than 515 nm using an additional cut-off filter (LP515) in front of the
detector. Fluorescence images were scanned and stored as a time series
(approx. 11 minutes). The peptides were applied to the cells 1 minute after
scanning began. Regions of interest (ROI's) were subsequently selected for
determination of the fluorescence intensities in the cytosol and the nucleus.
These data were stored as ASCII files and computed off-line. Different
solutions were used as the extracellular medium:
All three mediums (290 mOsm/kg) were adjusted to pH 7.4 (with NaOH).
Medium 1 represented a normal extracellular solution for intracellular
Ca2+ measurements (Weisner and Hagen, 1999). EGTA (in medium 2) has
a high affinity for Ca2+ and complexes the free Ca2+ in
the medium; under these conditions a Ca2+ influx into the cells is
impossible (Bers, 1998
;
McGuian et al., 1991
;
Miller and Smith, 1984
;
Williams and Fray, 1990
). The
function of cyclic nucleotide-gated (CNG) ion channels is blocked by
extracellular Mg2+ (three to five times more effectively than
Ca2+) (Frings et al.,
1995
; Weyland et al., 1994). With medium 3 we have the possibility
of testing whether the increase of intracellular Ca2+ concentration
is caused by a Ca2+ influx through such CNG channels. The
extracellular Mg2+ concentrations (10-15 mM) were selected so as
not to cause blockage, however, of voltage activated Ca2+ ion
channels (McDonald et al.,
1994
). The cells were variously preincubated for 30 minutes
with:
D-cis-diltiazem (Ferry et al.,
1993; Matlib and Schwartz,
1983
) and verapamil (Atlas and
Adler, 1981
; Fleckstein,
1977
) cause the blockage of voltage gated ion channels in the
plasma membrane. Staurosporine is an effective inhibitor of the cyclic
guanosine monophosphate (cGMP)- and the cAMP-dependent protein kinases, as
described previously (Matsumoto and
Sasaki, 1989
; Tamakori et al.,
1996
). These media and active compounds afforded various
possibilities of investigating changes in the intracellular Ca2+
signal.
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Results |
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|
|
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Characterization of the receptor-mediated Ca2+
response
The receptor antagonist -helical CRF(9-41) was used to clarify the
nature of the receptor-mediated signal pathway
(Chalmers et al., 1999
). Human
normal melanocytes were pre-incubated with 10-6 M antagonist for 20
minutes in cell medium, which was subsequently replaced by medium 1 for the
measurements. The pre-treatment with
-helical CRF(9-41) abolished or
significantly inhibited Ca2+ uptake in melanocytes after CRF or URC
stimulation (Fig. 3).
|
Measurements of intra-nuclear Ca2+ indicator
fluorescence
Intra-nuclear fluorescence intensity changes, measured after peptide
stimulation, are depicted in Fig.
4. The nuclear fluorescence values are expressed as ratios to the
corresponding cytosolic values. The data from HaCaT cells
(Fig. 4A) indicate a nuclear
Ca2+ fluorescence intensity 1.25-1.45-fold higher than that of the
cytosol after URC stimulation at 10-10-10-7 M, and a
ratio close to 1 at 10-13-10-11 M. In contrast, after
CRF stimulation, the ratios were consistently below 0.5 over the complete
concentration range employed, i.e. they were only approximately one-third as
high as those calculated for URC stimulation. This striking difference between
nuclear and cytosolic signals was not observed in the case of human malignant
melanocytes (Fig. 4B), in which
the median relative intensity increases were lower, e.g., at 1.3 after URC
(comparable to HaCaT cells), and 1.0 after CRF stimulation. Although at
peptide concentrations of 10-11-10-9 M the ratios were
significantly higher for URC than for CRF, these differences did not reach the
striking values observed for HaCaT keratinocytes. In addition, the ratios were
observed on neonatal epidermal keratinocytes and neonatal normal melanocytes.
These yielded median relative ratios (Fig.
4C) of 0.81 and 0.87, respectively, after CRF stimulation,
emphasizing again the differences between these cell lines and HaCaT
keratinocytes. Furthermore, at concentrations of 10-11 and
10-10 M of CRF the ratios were significantly lower in neonatal
keratinocytes than in neonatal melanocytes, indicating a difference between
epithelial and pigment cells. The time-gallery of confocal images
(Fig. 5) of HaCaT cells
confirms the effects shown in Fig. 6A and
B.
|
|
Time course of the changes of intracellular Ca2+
Fig. 5 illustrates the
variable intranuclear and cytosolic fluorescence changes following CRF and URC
stimulation. Thus, URC predominantly stimulates [Ca2+]i
flux into the nucleus (right panel), while CRF into the cytoplasm (left
panel). Note that there is an oscillatory effect of URC on
[Ca2+]i in HaCaT cells, while CRF induces a linear
accumulation of Ca2+. The relative fluorescence intensities
(cytosolic and intra-nuclear) from the images in
Fig. 5 over an 11 minute period
of observation are summarized in Fig. 6A
and B. Cytosolic fluorescence
(Fig. 6A) is seen to rise
continuously over the time period after CRF application, while that within the
nucleus follows only after an apparent lag of around 200 seconds. After 11
minutes, a significant increase in cytosolic fluorescence is established. The
signal pattern is, however, different after URC stimulation
(Fig. 6B). In this case, the
increase of intra-nuclear fluorescence intensity appears stronger from the
beginning than was indicated by the difference in relative intensities
(Fig. 4A). We have assumed that
calcium increases in the cytosol before reaching the nucleus; however, the
sensitivity of the time of measurements does not allow accurate accurate
assessment of intracellular localization.
Furthermore, URC-induced oscillations in Ca2+ levels are observed in both the cytosol and nucleus, as indicated in Fig. 5B. Additional data pertaining to these oscillations are presented in Fig. 6. It can be seen that the frequency of the oscillations is independent from the peptide concentration (Fig. 6C). It begins to change only at the concentration of 10-15 M. In addition to this concentration-independent variation in oscillation frequency, the numbers of cells displaying such oscillations remain rather constant with a decrease only at concentration of 10-15 M (Fig. 6D). The observed oscillatory behavior appears to be a specific characteristic of the response of HaCaT keratinocytes to URC stimulation, since it has not been seen in any other cell type.
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Discussion |
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The results with both peptides showed no correlation between peptide dosage
and increase of the fluorescence intensities (dose-effect-correlation). We
also observed the increase in intracellular Ca2+ levels at very low
peptide concentrations, e.g., 10-13 M (see Figs
1,
2), which contrasted with
reported KD's of 10-10-10-9 M and
10-8 M for CRF and URC in CHO cells transfected with CRF-R1 and
CRF-R2 cDNAs, respectively (Tojo and
Abou-Samra, 1993; Vaughan et
al., 1995
). This raised an important question on the nature of the
signal pathways activated by CRF and URC. The preincubation of the cells with
the specific CRF-R antagonist
-helical CRF(9-41) reduced CRF or URC
induced increases in the Ca2+ levels (see
Fig. 3). Moreover, previous
studies showed the expression of CRF-R1 in melanocytes and keratinocytes and
the presence of specific cell surface CRF binding sites
(Fazal et al., 1998
;
Slominski et al., 1995
;
Slominski et al., 1999
;
Slominski et al., 2000b
).
These receptors were functional, i.e. CRF and URC inhibited growth, stimulated
cAMP production and modulated the expression of adhesion molecules in cultured
human keratinocytes in a dose-dependent fashion
(Quevedo et al., 2001
;
Slominski et al., 2000b
;
Slominski et al., 2001
).
Therefore, we conclude that the reported stimulation of Ca2+
fluorescence intensity by CRF and URC is mediated through interaction with CRF
receptors. In other systems similar effects of CRF appear to be mediated by a
pathway involving the adenylate cyclase/cAMP-dependent protein kinase system
(Guild and Reisine, 1987
;
Reisine, 1989
;
Tojo and Abou-Samra,
1993
).
CRF-R1 is the most efficient receptor isoform for transducing the
CRF signal into cAMP mediated pathways, while other isoforms (ß, c and d)
are inefficiently coupled to cAMP production
(Chalmers et al., 1996
;
Linton et al., 2001
;
Perrin and Vale, 1999
; Speiss
et al., 1998). Since the most prevalent form of the CRF receptor expressed in
human skin is the R1
isoform
(Pisarchik and Slominski,
2001
; Slominski et al.,
2001
), and STA significantly inhibits the stimulatory effect of
CRF on Ca2+, we suggest that some of these effects are mediated by
CRF-R1
through cAMP-dependent protein kinases. However, the
voltage-gated ion channel blockers DIL and VER also block Ca2+
influx. The stimulatory effect is observed at ligand concentration
10-13 M, i.e. far below the KD's described for CRF-R1
(see above). The last finding is consistent with previously reported data
showing that CRF at 10-12 M can induce entry of Ca2+ in
melanoma cells (Fazal et al.,
1998
). However, the reported effect was very rapid (within one
second), while the current changes were observed only after several minutes of
incubation. Referring to the time response similar Ca2+ signals
were observed after ACh-induced Ca2+ release in oocytes expressing
two hybrid mAChRs (Lechleiter, 1991). In this investigation a continuous
increase in the Ca2+ signal around 500 seconds after stimulation
was found. As regards to the response to low peptide concentrations another
study showed that CRF-like peptides 10-15 M stimulated a
Ca2+ response in human epidermoid A-431 cells
(Kiang, 1997
). Moreover,
stimulus-response curves for muscarinic and
-adrenoreceptors indicate
that maximal response can be produced by submaximal stimuli (submaximal
receptor occupancy) (Kenakin,
1993
). This phenomenon, described as "receptor
reserve" (spare receptors, spare capacity), leads to an ultrasensitivity
of the system. In this case, the dose-response curve is not a typical
Michaelis-Menten curve but rather a nearly digital response (all or nothing)
(Germain, 2001
).
Therefore, we suggest that skin cells co-express different CRF-R forms,
which are coupled to different signal transduction pathways
(Pisarchik and Slominski,
2001; Slominski et al.,
2001
). Although the individual functions of the various receptor
subtypes remains to be determined, the differential regulation of
Ca2+ signal by CRF and URC in HaCaT keratinocytes (Figs
5,
6) show similarities to those
described by others (Germain,
2001
; Kenakin,
1993
; Kiang, 1997
;
Lechleiter et al., 1991
;
Reetz and Reiser, 1996
).
Unusual ligand induced Ca2+ signaling in HaCaT
keratinocytes
The strong signal enhancement observed in the nucleus of HaCaT cells after
stimulation with URC is in striking contrast with the absence of such an
effect after exposure to CRF. It has previously been accepted that the
intracellular signal pathway is the same for both peptides. The existence of
Ca2+-permeable pores in the nuclear membrane is indisputable
(Bhattacharya et al., 1999;
Kenakin, 1993
), but we
observed Ca2+ flux into the nucleus only in the case of URC.
IP3 receptors have also been reported on the nuclear membrane. When
IP3 binds to these receptors, a Ca2+ influx from the
cytosol to the nucleus, or conversely, Ca2+ release from the
nucleus in the opposite direction are possible (Challis and Wilcox, 1996;
Wilcox et al., 1994
). In this
case, an increase of intracellular IP3 would be expected after
stimulation with URC. Although IP3 signaling has not been analyzed
in HaCaT cells, CRF-related peptides induce IP3 production and
subsequent Ca2+ mobilization in A-431 epidermal carcinoma cells
(Kiang et al., 1995
).
Therefore, it is likely that in HaCaT keratinocytes the URC signal would be
similarly coupled to production of IP3. In addition, these data
show that CRF and URC signals are transduced through different receptor
subtypes in keratinocytes. Furthermore, the nuclear targeting of the URC
signal (e.g., intracellular calcium oscillations) suggests a role for this
pathway in transcriptional regulation. Others also reported nuclear calcium
transient rise in response to integrin-ligand interaction
(Shankar et al., 1993
) or
after stimulation of rat liver nuclear prostaglandin receptors
(Bhattacharya et al.,
1999
).
The differences in time courses of the fluorescence signals after peptide
stimulation are intriguing. Stimulation of HaCaT cells with URC produced a
remarkable oscillatory effect. Oscillation of intracellular Ca2+
levels was previously demonstrated in bovine pulmonary artery endothelial
cells over approximately 15 minutes after stimulation with the peptide
bradykinin (Sage et al.,
1989). A similar oscillatory increase in cytosolic Ca2+
concentration was observed after stimulation with CRF
(Kuryshev et al., 1996
).
Furthermore, a long-term perfusion with bradykinin was described as causing
oscillation of cytosolic Ca2+ activity in rat glioma cells
(Reetz and Reiser, 1996
). This
Ca2+ rise was associated with synchronous plasma membrane
oscillating hyperpolarization. The authors proposed that the initial transient
Ca2+ rise induced immediately with bradykinin administration
resulted from IP3-mediated Ca2+ release, whereas
subsequent oscillations depended mainly on Ca2+ influx. Such
Ca2+ spikes were observed in time intervals from seconds to
minutes. This phenomenon was generally discussed as a mechanism of
regeneration, e.g. Ca2+influx stimulates an increase in
phospholipase C activity whereby an increase of IP3 occurs and this
results in a Ca2+ release
(Woods et al., 1986
).
Oscillating Ca2+ concentrations might thus have been anticipated
after stimulation of CRF receptors. Our data indicate, however, that this was
the case only after stimulation with URC i.e. not after CRF stimulation (see
Fig. 6). Thus, these results
show that HaCaT keratinocytes behave distinctly differently from other cell
types and imply the presence of an intracellular signal transduction pathway
for URC. In this context, HaCaT keratinocytes serve as an excellent cell model
for further characterization of this pathway.
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Acknowledgments |
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