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Divisions of Nephrology and Endocrinology, Department of Medicine, Cedars-Sinai Research Institute, Cedars-Sinai Medical Center, University of California School of Medicine, Los Angeles, California 90048
Address all correspondence and requests for reprints to: Jacob Green, M.D., Department of Nephrology, Rambam Medical Center, Haifa 31096, Israel. E-mail: greeny{at}rambam.health.gov.il
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study was undertaken to determine the effect of ECM constituents on PTH binding and PTH responsiveness (cAMP generation) in the OK opossum kidney cell line, which manifests many proximal tubular features (7). Because one of the cAMP-mediated effects of PTH is the modulation of the Na+/H+ exchanger (NHE) in the proximal tubule (8), we also studied the effect of various matrices on the exchanger in the OK cells, with and without PTH. We have recently shown (9) that, in osteoblastic bone cells, generation of cAMP by PTH is augmented in cells exposed to collagen. We therefore compared results obtained in the epithelial kidney cells to those obtained in the mesenchymally derived MC3T3-E1 cells, a clonal line of nontransformed murine osteoblasts (10). Our results show divergent effects of cell-matrix interaction on PTH responsiveness between the two cell types.
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Materials and Methods |
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Cell culture
OK cells were obtained as a gift from Dr. D. Warnock of
Birmingham, AL, and were used at passages 30–55. The cells were grown
in Ham’s F-12 + DMEM (1:1) supplemented with 14.3 mM
NaHCO3, 1.2 mM L-glutamine, 10%
FBS, 50 U/ml penicillin, and 50 µg/ml streptomycin. Cultures were
maintained at 37 C in 5% CO2. Cells were subcultured
weekly using 0.1% trypsin and were plated at a cell density of 5
x 104 cells/cm2. Plating was done onto plastic
or tissue culture dishes (multiwell plates, coverslips) coated with
Col-IV, laminin, or fibronectin. The cells became confluent after
72 h, and experiments were done on day 4 of culture. Unless
otherwise indicated, the cells were first cultured in medium
supplemented with FBS, before switching to serum-free conditions.
MC3T3-E1 cells were cultured in
amino acid-free Modified Eagle’s Medium (MEM-
) and 10% FCS
containing 100 U/ml penicillin and streptomycin and passaged every 3–4
days. The cells were plated at an initial density of 25,000
cells/cm2 on plastic or tissue culture dishes coated with
Col-IV, fibronectin, or laminin. Experiments were done on day 4 of
culture.
Determination of cellular cAMP levels
Determination of cellular cAMP levels was done in culture plates
containing 24 multiwells/plate. Cells grown on plastic or various ECM
matrices were acutely (from 5 min to 2 h) stimulated with agonists
dissolved in 1 ml balanced salt solution at 37 C in the presence or
absence of 0.2 mM 3-isobutyl-1-methylxanthine. The reaction
was terminated by aspirating the medium containing the stimulant and
then adding 0.5 ml L-propanol to extract the cAMP from the
cell layer. The cell layers were then kept at 4 C for 1 h. The
propanol extract was removed to glass tubes, the propanol was
evaporated under a stream of nitrogen gas, and the dried extract was
kept at -70 C until assay. Before assay, the extract was reconstituted
with sodium acetate buffer, pH 6.2. Assay of cAMP was carried out by
RIA with minor modification. [125I] Succinyltyrosine
ester of cAMP (ICN, Irvine, CA; 10,000 cpm/100 µl) was used.
Antigen-antibody precipitation was done by 100% ethanol. Results are
expressed as pmol cAMP/106 cells.
PTH/PTHrP receptor binding studies
[Tyr36] human PTHrP-(1–36) NH2 was
radioiodinated and purified by HPLC to a final estimated specific
activity of 2,200 Ci/mmol. Binding studies were carried out in
24-multiwell dishes containing confluent
MC3T3-E1 or OK cells. Binding of
radioligand was performed by removing the medium and incubating the
cells at 15 C in binding buffer [50 mM Tris-HCl (pH 7.5),
100 mM NaCl, 2 mM CaCl2, 5
mM KCl, 0.5% heat-inactivated BSA, and 20% FBS]
containing 250,000 cpm tracer in the presence or absence of different
concentrations of unlabeled PTHrP-(1–34) NH2. Binding
reactions were terminated by aspirating the buffer and washing the
monolayers three times with ice-cold 0.9% NaCl. Cells were then
treated with 200 µl 1.0 N NaOH and transferred to test
tubes. Cell-associated radioactivity was determined by
-counting.
Total RNA extraction and Northern blot analysis
Total RNA was extracted according to the method of Chirgwin
et al. (11), with slight modifications. Fifty micrograms of
total RNA (from two confluent T-75 flasks) were separated
electrophoretically on 1.5% agarose, formaldehyde gels containing
ethidium bromide. RNA samples were transferred onto nylon membranes and
then hybridized to a PTH/PTHrP receptor complementary DNA spanning
nucleotides 1–1810 labeled with [32P]cytidine
5'-triphate by a random primer method. Filters were washed three times
(30 min each) to a stringency of 0.1x sodium citrate, 0.1% SDS at 55
C, and exposed to Kodak X-omat film (Eastman Kodak, Rochester, NY) with
intensifying screens at -70 C.
Measurements of intracellular pH (pHi)
Cells grown to confluence on coverslips coated with different
ECM components were washed and suspended in a balanced salt solution
containing (in mM) 140 NaCl, 1 MgCl2, 4 KCl, 10
HEPES-Tris (hydroxymethyl) aminomethane, 1.5 CaCl2, 5
glucose, and 5 sodium pyruvate, pH 7.4 (adjusted with 1 M
NaOH). The cells were then loaded with BCECF by incubation at 37 C with
2 µM of its cell permeant tetraacetoxymethyl ester (AM)
for 15 min. BCECF-AM enters the cells, and cytoplasmic esterases
convert it to free BCECF, the pH-sensitive form. The cells were then
washed once and kept in the same medium at room temperature until
use.
For fluorescence recording, the coverslips were mounted in a perfusion chamber and continuously perfused. The perfusate volume in the chamber was adjusted to 0.3 ml, and the cells were perfused at 10–12 ml/min. The perfusate was delivered through an eight-way valve to a heat exchanger, then to the chamber, and maintained at 37 C. The recording system included a Nikon Diaphot inverted microscope equipped with a high numerical aperture Neofluor x 100/1.3 numerical aperture (Carl Zeiss, Aalen, Germany) oil-immersion objective. The microscope was attached to a Photon Technology International Delta Scan spectrofluorometer, which provided a dual-wavelength excitation light of 450 and 500 nm. The excitation light was selected by a spinning chopper mirror and directed to the cell by a dichroic mirror. The emitted light of 530-nm wavelength was monitored by a photomultiplier tube at a resolution of 3/sec, and the signal was stored in an NEC Power Mate 1 computer for further analysis. pHi was obtained from the uncorrected ratios of 500/450 after appropriate calibration, as described by Thomas et al. (12).
To study the activity of the NHE, we measured the recovery of the cell from an acid load using the NH4Cl pulse technique (13). Briefly, cells were loaded with BCECF while being exposed for 20 min to a solution containing (in mM) 125 NaCl, 20 NH4Cl, 5 KCl, 5 HEPES, 1 CaCl2, 0.5 MgCl2, and 10 glucose (pH 7.4). After a wash of the cells with the same solution (to remove extracellular dye), the cells were resuspended in Na+-free buffer (140 mM tetramethylammonium chloride) with the rest as above, but without NH4Cl. The removal of NH4Cl results in rapid acidification of the cell. Finally, the cells were perfused with NaCl solution. The Na+-dependent pHi recovery rate was measured with and without amiloride. The initial 30-sec recovery rate was used to estimate the pHi recovery rate.
Determination of cell proliferation
Cell proliferation was assessed by the incorporation of
[3H] thymidine. Briefly, cells were grown in 24-well
plates 22 h before experimentation; the medium was changed to
serum-free Ham’s F-12-DMEM. Three hours before the harvest, cells were
pulsed with 0.2 µCi/ml [3H] thymidine (6.7 Ci/mmol).
Cells were harvested by three PBS washes to remove unincorporated
label, followed by two washes with 10% trichloroacetic acid. The cell
layers were solubilized in 1 N NaOH, and aliquots of the
solubilized cells were diluted into liquid scintillation fluid after
neutralization with HCl and were counted in a ß-counter. Cells were
counted by hemacytometer, and data were expressed as
cpm/106 cells.
Statistics
Results are expressed as means ± SD. Nonlinear
square curve fitting was used to assess dose-response curves to
estimate ED50 and maximally effective concentrations of
agonist with 67% confidence limits, assuming highly correlated
asymmetric variance spaces. One- and two-way ANOVAs for differences
among treatment means were performed, as indicated, where appropriate.
Each experiment was performed at least four times with separate batches
of cells to confirm reproducibility of the results.
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Results |
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shows that,
in both renal proximal tubular cells (OKP) (right panels)
and the MC3T3-E1 osteoblastic cells
(left panels), there was a marked reduction (approximately
50%) of cAMP production stimulated by 1–34 PTH (10-8
M) when cells were plated on Col-IV, compared with cells
plated on plastic (P < 0.001). Col-IV did not affect
cAMP production stimulated by PGE2, nor did it have any
effect on postreceptor mechanisms for cAMP production. Thus, the cAMP
response to cholera toxin (which activates the stimulatory subunit of
adenylyl cyclase) and forskolin (which activates the catalytic subunit
of adenylyl cyclase) was not different in cells plated on Col-IV, when
compared with cells on plastic. The suppressive effect of Col-IV on
cAMP production mediated by PTH could not be ascribed to modified
activity of phosphodiesterase, because the same qualitative effect was
observed both in the absence (Fig. 1, lower panels, B) or
the presence (Fig. 1, upper panels, A) of the
phosphodiesterase inhibitor isobutyl-L-methylxanthine
(IBMX). Also, the effect of Col-IV was not abolished after
preincubation of the cells with pertussis toxin (100 ng/ml for 24
h), suggesting that the inhibition of PTH-mediated cAMP production was
not related to activation of the inhibitory guanine nucleotide binding
protein (Gi) (data not shown).
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As shown in Fig. 2, the effect of Col-IV
on cAMP production by PTH (10-8 M) was dose
(density) dependent. In both cell types, P < 0.01 for
plastic vs. low-density Col-IV (50 µg/ml), and
P < 0.001 for plastic vs. high density
Col-IV (500 µg/ml.
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). In
MC3T3-E1 cells, the first
significant effect was already observed after 6 h of plating on
Col-IV (P < 0.01). However, in OK cells, the first
significant effect was observed only after 12 h of plating on
Col-IV (P < 0.01).
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The effect of Col-IV on PTH/PTHrP binding
As shown in Fig. 4, high-affinity
binding of [125I]PTHrP was demonstrated in both the
kidney and osteoblastic cells. The radioligand displacement curve shows
reduced hormonal binding in both cell types when plated on Col-IV
vs. plastic. Scatchard plot analysis (bottom part
of figure) yielded the following values: in
MC3T3-E1 cells, Kd =
8.42 ± 0.12 nM, and 8.35 ± 0.1 nM
in plastic and Col-IV, respectively (no significance). Maximum sites
per cell (Bmax) (sites per cell x 105) =
2.15 ± 0.2 and 1.15 ± 0.2 in plastic and Col-IV,
respectively (P < 0.01). Corresponding values in OK
cells showed the following: Kd = 1.7 ± 0.06 and
1.82 ± 0.07 nM in cells on plastic and Col-IV,
respectively (no significance). Bmax (sites per cell
x 105) = 1.8 ± 0.1 and 0.95 ± 0.15 in cells on
plastic and Col-IV, respectively (P < 0.01).
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, the PTH/PTHrP
receptor mRNA transcript was clearly detected in both OK and
MC3T3-E1 cells. However, the
receptor expression is approximately 4- to 5-fold greater in OK cells
vs. MC3T3-E1 cells. The
expression of the receptor was examined in each cell type grown on
plastic, Col-I, or Col-IV. In OK cells, the relative abundance of the
PTH/PTHrP receptor mRNA was reduced in cells grown on Col-IV, compared
with cells on plastic. In cells grown on Col-I, the receptor expression
was similar to that observed under control conditions (cells on
plastic). Densitometry measurements were performed on the Northern
blots exposed for 2 or 4 h. The following results (peak weights)
were obtained: at 2 h: control (plastic) = 0.1658, Col-I =
0.1565, Col = IV-0.0913; at 4 h: control = 0.2353,
Col-I = 0.2132, Col-IV = 0.1255. Thus, at both time points,
there was roughly 53–55% reduction in PTH/PTHrP receptor mRNA by
Col-IV.
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The physiological significance of the Col-IV effect on cAMP
production by PTH
Because a Col-IV matrix attenuates the production of cAMP by PTH
in a specific manner, it is conceivable that those biological functions
of PTH that are mediated by cAMP would be modified in cells exposed to
excess Col-IV. To explore this notion further, we decided to examine
the effect of various ECM components on two cAMP-mediated biological
functions of PTH: 1) the activity of Na+/H+
antiporter (exchanger) in the kidney OK proximal tubular cells; and 2)
the effect of PTH on cell proliferation in
MC3T3-E1 cells.
NHE and ECM
The amiloride sensitive, electroneutral NHE is an ubiquitous
transporter that serves multiple functions, including pHi regulation,
volume regulation, and promotion of cell growth (15). In OK cells, the
NHE is segregated in the apical cell membrane, and it is the main
determinant of steady-state pHi in these cells (16); it is also the
major mechanism for apical proton secretion and NaHCO3
reabsorption (reclamation) in the proximal tubule (17). The activity of
the exchanger is modulated by a variety of hormones and second
messengers; PTH inhibits the activity of NHE, an effect mediated by
cAMP (18). With this background in mind, we studied the effect of ECM
components on NHE activity, with and without exposure to PTH.
Others and we have shown (16, 19) that, in HEPES-buffered medium,
recovery from an acid load in OK cells is largely mediated by the NHE.
Figure 6 shows the pHi recovery pattern
after cell acidification, by the NH4Cl pulse technique
(13), under control conditions (cells on plastic) and in cells plated
on Col-I or Col-IV. Under control conditions, pHi recovered at a rate
of 0.42 ± 0.03 
pH/min. This response was 95% blocked by 0.1
mM amiloride, an inhibitor of the NHE. Also, the
alkalinization response was Na+-dependent because, in the
absence of Na+ in the media, only minimal recovery from the
acid load was observed (not shown). Thus, the recovery of pHi, under
these conditions, is sodium dependent and amiloride sensitive,
indicating that this response is mediated by the NHE.
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pH/min = 0.12 ± 0.01, P <
0.001 vs. plastic). Col-I matrix also inhibited NHE
activity, albeit to a lesser degree than Col-IV ( pH/min =
0.31 ± 0.04, P < 0.05 vs.
plastic).
Figure 7 shows the pattern of NHE
activities in cells plated on plastic (control) or Col-IV. In addition,
the cells were acutely (approximately 20 min) stimulated with either
1–34 bPTH (10-8 M) or with vehicle. Cells on
Col-IV (without PTH) again manifested the marked inhibition of
exchanger activity, when compared with cells on plastic. In cells grown
on plastic (control), acute exposure to PTH resulted in an inhibition
of NHE ( pH/min = 0.46 ± 0.04 vs. 0.32 ±
0.02 cells on plastic without PTH and cells exposed to PTH,
respectively, P < 0.05). In cells grown on Col-IV,
acute exposure to PTH had an opposite response; namely, stimulation of
the exchanger by approximately 130%, when compared with NHE activity
in cells plated on Col-IV and exposed to vehicle alone ( pH/min
= 0.12 ± 0.01 vs. 0.28 ± 0.04, cells on Col-IV
without PTH and cells exposed to PTH, respectively, P
< 0.01).
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shows a dose-dependent
antiproliferative effect of PTH in the osteoblast-like
MC3T3-E1 cells. When compared with
cells on plastic, cells grown on Col-IV manifested a marked reduction
in the antiproliferative effect of PTH (40–50% difference),
consistent with the notion that decreased cAMP production by PTH, in
cells grown on Col-IV, mitigates the antiproliferative effect of PTH.
Col-IV, in the absence of exposure to PTH, did not have an effect of
its own on cell proliferation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Aminoterminal analogs of PTHrP had a cAMP response similar to that of PTH(1–34), when tested in both kidney and bone cells plated on plastic. Moreover, Col-IV had the same suppressive effect on cAMP production induced by these peptides, as seen with PTH(1–34). These effects were not reproduced with C-terminal and midregion PTHrP fragments in either cell type. These findings are in accord with other investigators, showing that the PTH-like N-terminus is necessary for activation of second-messenger pathways in different types of cell systems (22).
The hyporesponsiveness to PTH and PTHrP at the receptor level could result from one or more of the following: internalization and degradation of the receptor, phosphorylation of the receptor or its coupled G protein, or reduced expression of receptor mRNA followed by reduced protein synthesis. Our study indicates that, in both kidney and bone cells, Col-IV inhibits PTH receptor binding caused by a reduced number of receptors. However, the response of the kidney cells differed from that of osteoblasts, in that the attenuation of cAMP production is MC3T3-E1 cells was first observed when the cells were plated on Col-IV for 6 h, whereas in the OK proximal tubular cells, the first significant effect was observed only after 12 h of plating on Col-IV. The different time course between the two cell types may be linked to the fact that, in the OK cells, Col-IV induced an attenuation of the steady-state level of the PTH/PTHrP receptor mRNA, whereas such an effect could not be observed in the osteoblastic cells. It seems, therefore, that the phenomenon of reduced PTH receptor number by Col-IV could be related to either transcriptional (OK cells) or posttranscriptional (MC3T3-E1 cells) mechanisms. The posttranscriptional event could involve internalization and degradation of the receptor. Indeed, an effect of collagen matrices on receptor internalization and degradation has been shown in glomerular epithelial cells [for the epidermal growth factor receptor (23)] and in fibroblasts [for platelet derived growth factor receptor (24)].
Regardless of the cause of the reduced PTH receptor expression (i.e. genomic vs. nongenomic), our results are in accord with other studies (25) showing that, in LLC-PK renal epithelial cells transfected with the rat and opossum PTH/PTHrP receptor complementary DNA, the magnitude of cAMP response to PTH is correlated with the actual number of receptors. Thus, the reduced number of PTH receptors, in OK and MC3T3-E1 cells plated on Col-IV vs. plastic substrata, could account for the attenuation of cAMP response by Col-IV in both cells types.
Our study adds to a growing body of evidence showing that adhesion of
cells to ECM influences cell growth and differentiation and can lead to
modulation of intracellular signal transduction pathways activated by
hormones (1). An effect of ECM proteins on signal transduction
mechanisms has been demonstrated in many cell types, including kidney
and bone cells, the two cell systems used in our study (9, 26, 27).
Attachment of cells to ECM is mediated through cell surface adhesion
receptors, many of which belong to the family of heterodimeric
transmembrane glycoproteins known as integrins (1, 2). Cell surface
integrins comprise two noncovalently linked chains ( and ß); and
because of the large number of ß combinations, the number of
integrins has been steadily increasing. Some of the ß1
integrins are considered to be classic collagen receptors
(e.g. 1 ß1,
2ß1, 3ß1) and
have been shown to affect biological processes in kidney and bone
cells, both under normal and pathological states (28, 29). Based on
this information, we assume that one or more of the ß1
integrins are involved in the modulation of hormonal responses observed
in our study. It is therefore hypothesized that binding of the integrin
receptors on kidney and bone cells to the Col-IV substratum is followed
by reorganization of the cell cytoskeleton, which then leads to genomic
(in kidney cells) and nongenomic effects (in bone cells) on the PTH
receptor.
The physiological significance of our studies is demonstrated in both the OK cells and the MC3T3-E1 cells. In the kidney proximal tubular cells, plating on Col-IV brings about marked attenuation of the NHE activity. This effect is observed, albeit to a lesser degree, with Col-I as well. When the effect of PTH on the exchanger is tested, one finds an inhibitory effect of PTH on NHE when cells are plated on plastic, consistent with results generated by many other investigators (18). Quite remarkably, PTH shows an opposite effect when cells are plated on Col-IV, namely, marked stimulation of the NHE. Inasmuch as the inhibitory effect of PTH on NHE is mediated by cAMP, it is assumed that the inhibition of cAMP production in cells on Col-IV removes an inhibitory force affecting the exchanger; and therefore, the final outcome is stimulation, rather than inhibition, of NHE in cells grown on Col-IV. This effect of PTH is reminiscent of the stimulatory effect of angiotensin II on the proximal tubular NHE, which is also related to the inhibition of cAMP (30). Given the cardinal effect of NHE on proximal Na+ reabsorption (17), it is conceivable that the in vitro modulation of NHE is translated into in vivo alteration in Na+ retention along the proximal tubule. This notion may be of relevance in at least two pathological states; namely, diabetes mellitus and glomerulonephritis. Both diseases are characterized by ECM expansion (29, 31); and therefore, the tubular cell responsiveness to PTH may be switched from a salt-losing to a salt-retention mode. Indeed, salt-sensitive hypertension is a common observation in both diabetes and glomerulonephritis.
With regard to osteoblasts, the biological significance of the Col-IV effect on PTH-mediated cAMP production may by linked to the fact that, under physiological conditions, cAMP exerts an antiproliferative effect in osteoblasts (21). Thus, in cells plated on Col-IV, compared with cells on plastic, the antiproliferative effect of PTH was attenuated, consistent with diminished cAMP production under these circumstances. Moreover, the cAMP signaling pathway in osteoblasts has been demonstrated by many investigators to have an antianabolic effect in bone and to serve as a bone-resorption signal (20, 32). We speculate that, by down-regulating PTH-mediated cAMP production in cells exposed to Col-IV, bone resorption processes may be modulated in a negative direction (i.e. diminished bone resorption).
In conclusion, this study illustrates events related to modulation of signal transduction pathways brought about by cell-matrix interaction in kidney and bone cells. Interstitial and BM collagens are produced and secreted by renal tubular cells, as well as by osteoblasts, and therefore can influence hormonal responsiveness, in an autocrine fashion. This phenomenon may be of relevance under normal conditions, as well as in disease states (e.g. diabetic nephropathy, tubulointerstitial diseases of the kidney). Furthermore, on the basis of this study, it is clear that, while studying cell signaling by using in vitro culture systems of bone and kidney cells, one needs to take into consideration the type of substratum onto which the cells are plated.
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Acknowledgments |
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Footnotes |
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2 Present address: Division of Endocrinology and Metabolism,
University of Cincinnati, College of Medicine, Cincinnati, Ohio
45267.
Received December 9, 1997.
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and V. Kidney Int 52:103–110[Medline]
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