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Endocrinology Vol. 139, No. 3 1023-1030
Copyright © 1998 by The Endocrine Society

ARTICLES

Differential Expression of Calcitonin and Parathyroid Hormone/Parathyroid Hormone-Related Protein Receptors in P19 Embryonic Carcinoma Cells Treated with Retinoic Acid1

Marianne Eggenberger, R. Anne McKinney, Jan A. Fischer and Roman Muff

Research Laboratory for Calcium Metabolism, Departments of Orthopedic Surgery and Medicine (M.E., J.A.F., R.M.), Brain Research Institute (R.A.M.), University of Zurich, 8008 Zurich, Switzerland

Address all correspondence and requests for reprints to: Dr. R. Muff, Klinik Balgrist, Forchstrasse 340, 8008 Zurich, Switzerland. E-mail: ramuz{at}balgrist.unizh.ch


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Mouse embryonic carcinoma P19 cell aggregates treated with retinoic acid (RA) sequentially differentiate into neurons and astrocytes, whereas attached cells develop a mesodermal phenotype. The expression of calcitonin (CT) and PTH/PTH-related protein (PTHrP) receptors was investigated in embryonic cells, and during neural and mesodermal differentiation. In embryonic P19 cells, specific binding of [125I]salmon (s) CT(1–32) ([125I]sCT(1–32)) was 56 fmol/mg protein, and of [125I]chicken (ch) [Tyr36]PTHrP(1–36) amide ([125I]-chPTHrP(1–36)) < 0.5 fmol/mg protein. Correspondingly, cAMP was maximally stimulated 47-fold by sCT(1–32) (EC50 0.05 nM) and 3-fold by chPTHrP(1–36) (EC50 1.3 nM). Receptor autoradiography revealed specific binding of [125I]sCT(1–32) to the undifferentiated P19 cells, but not to RA induced neurons and astrocytes. At the same time, [125I]sCT(1–32) binding and cAMP accumulation by sCT were gradually decreased. But, specific binding of [125I]chPTHrP(1–36) was raised at least 6-fold compared with embryonic cells to 3 fmol/mg protein, in parallel with a 10-fold higher maximal cAMP accumulation. A similar, but delayed suppression of CT and stimulation of PTH/PTHrP receptor expression was observed during mesodermal cell differentiation. The results indicate that CT receptors are associated with undifferentiated P19 cells, whereas PTH/PTHrP receptors are expressed in RA induced neural and mesodermal cells.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CALCITONIN (CT) receptors have been recognized in different tissues, such as bone, kidney and in the central nervous system (1, 2). Calcitonin is known to enhance cAMP accumulation in rat brain cells in primary culture (3). Intracerebroventricular injection of CT inhibits pain perception and feeding in rats and increases PRL secretion (1). CT-like peptides have been identified in human and rat brains (1, 4).

PTH and PTH-related protein (PTHrP) interact with the same PTH/PTHrP receptor identified in kidney, bone, and brain (5, 6). To this end, PTH and PTHrP raise cAMP in cultured rat astrocytes and cytosolic free calcium in hippocampal neurons (3, 7, 8, 9). Biological actions of intracerebroventricularly administered PTH include hyperalgesia, impairment of learning and memory processes and stimulation of PRL secretion (for review, see Ref.10). PTH and PTHrP-like peptides, and a PTH/PTHrP receptor are widely distributed in the central nervous system (11, 12, 13, 14). Another receptor, the PTH2 receptor, cloned from a rat cerebral cortex complementary DNA library, is recognized by PTH, but not by PTHrP, and by a PTH2 receptor selective ligand distinct from PTH, isolated from the hypothalamus (15, 16). Recently, a distinct receptor, which recognizes PTHrP unlike PTH, has been identified in the rat supraoptic nucleus (17).

Upon treatment with retinoic acid (RA) mouse embryonic carcinoma P19 cells differentiate into neural and mesodermal cell types (18). Neural differentiation is obtained with RA in aggregated cells with extensive contacts and the developing neurons and astrocytes are identified morphologically and immunohistochemically (19, 20). In the absence of cell contacts mesodermal cell types are developed in response to RA.

In the present study, the differential expression of CT and PTH/PTHrP receptors has been examined in embryonic carcinoma P19 cells both before and after differentiation into neurons, astrocytes, and fibroblast-like cells of mesodermal origin. Specific binding of [125I]salmon (s) CT(1–32) was limited to embryonic cells and was gradually reduced during neural and mesodermal differentiation. PTH/PTHrP receptors, on the other hand, became recognizable irrespective of the differentiation pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture
Mouse embryonic carcinoma P19 cells, obtained from the American Type Culture Collection (Rockville, MD; ATCC CRL 1825), were cultured in a humidified atmosphere of 5% CO2 and 95% air at 37 C in DMEM/Ham F12 (1:1) supplemented with 10% heat inactivated FCS, 2 mM glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. The cells were subcultured every 3–4 days with 0.1% trypsin/0.5 mM EDTA (trypsin/EDTA) in PBS (150 mM NaCl, pH 7.4).

To induce neural differentiation (18), trypsinized P19 cells were aggregated at a density of 100,000 cells/ml in bacteriological grade petri dishes (Greiner GmbH, Frickenhausen, Germany) in the presence of 1 µM all-trans retinoic acid (RA; Sigma, St. Louis, MO). The medium was renewed after 2 days. On day 4, the aggregates were plated in the absence of RA into either 24-well plates or onto poly-L-lysine (0.05 mg/ml) coated coverslips. For receptor autoradiography, aggregates were dissociated with trypsin/EDTA on day 4 and seeded into slide flasks (Nunc, Roskilde, Denmark). Days in culture include the 4 days RA treatment period.

To induce mesodermal differentiation (18, 19), P19 cells were seeded into tissue culture flasks (4000 cells/cm2) and treated with 1 µM RA. The medium was renewed after 2 days. On day 4, the cells were removed from the culture flasks with trypsin/EDTA and seeded into 24-well plates in the absence of RA.

Immunocytochemistry
Control P19 and cells cultured for 6–14 days, grown on coverslips, were rinsed with PBS before fixing in buffered 4% paraformaldehyde at room temperature for 30 min. After fixation, the cultures were washed in PBS and then preincubated in PBS containing 0.1% Triton X-100 and 2% horse serum at room temperature for 10 min. Cells cultured for 18–30 days were treated in a similar manner but the preincubation time was increased to 90 min as more cells were present. After the permeabilization step, the cells were washed with PBS before incubation at 4 C overnight with the appropriate primary antibody either antimouse IgG Neurofilament NF160 kDa (1:40 dilution in PBS containing 2% horse serum) or antimouse IgG glial fibrillary acidic protein (1:40 dilution in PBS containing 2% horse serum) (Boehringer Mannheim, Germany). The primary antibody was revealed using the direct immunocytochemistry method with fluorescein isothiocyanate (DTAF) conjugated goat antimouse IgG (Jackson Immuno Research Laboratories Inc., West Grove, PA) (1:100 dilution in PBS containing 2% horse serum)) at room temperature for 4 h. The cultures were washed three times with PBS and once with demineralized water, mounted in glycerol and examined with a Zeiss Axiophot microscope equipped with a Neofluar objective (40 x; 0.75 numerical aperture (N.A.); Carl Zeiss, Oberkochen, Germany).

Receptor autoradiography and binding studies
[125I]sCT(1–32) and [125I, Tyr36]chicken (ch) PTHrP(1–36) amide ([125I]chPTHrP(1–36)), radiolabeled by the chloramine-T method and purified by reverse phase HPLC, had a specific radioactivity of 2000 Ci/mmol (21, 22).

For receptor autoradiography the cells were grown in slide flasks and washed once with DMEM/Ham F12 (1:1) supplemented with 0.1% BSA. The slides were incubated at 15 C in 1 ml with 1.25 nM [125I]sCT(1–32) for 2 h. The slides were rinsed with PBS, and the cells fixed with 4% paraformaldehyde in PBS at room temperature for 15 min. The slides were then washed with PBS and demineralized water, coated with 1% gelatine and dipped into a photoemulsion diluted 1:2 in 0.5% glycerol (NTB2; Scientific Imaging Systems, Eastman Kodak Company, New Haven, CT) and exposed at 4 C for 7 days. Nonspecific binding was examined in the presence of 1 µM sCT(1–32). After development, the cells were visualized with a Nikon inverted microscope with a plan objective (10 x; 0.3 N.A.; Diaphot TMD, Nikon Corporation, Tokyo, Japan).

Receptor binding of 0.125 nM [125I]sCT(1–32) and 0.125 nM [125I]chPTHrP(1–36) and displacement by sCT(1–32), chPTHrP(1–36) and rat (r)PTH(1–34) (donated by S. Guttmann, H. Rink, and E. Felder, Novartis, Basel, Switzerland) was carried out in 24-well plates in 200 µl of the medium used for receptor autoradiography. For saturation binding experiments, cells were incubated with increasing amounts of radioligand in the absence and presence of 1 µM unlabeled peptide. After incubation at 15 C for 2 h the cells were rinsed once with ligand-free incubation medium. After solubilization of the cells with 500 µl 0.5% SDS, radioactivity was measured with a MR252 {gamma}-counter (Kontron, Switzerland). Specific binding is defined as the difference between total binding and the binding in the presence of 1 µM unlabeled sCT(1–32) or chPTHrP(1–36).

Cellular cAMP accumulation and determination of protein
Cells grown in 24-well plates were incubated in 200 µl 136 mM NaCl, 5.5 mM glucose, 5.4 mM KCl, 1 mM Na2HPO4, 1 mM CaCl2, 1 mM MgSO4, 1 mM 3-isobutyl-1-methylxanthine, 20 mM HEPES, pH 7.45 and 0.1% BSA at 37 C for 15 min. The medium was removed and cellular cAMP was extracted with 500 µl 95% ethanol, pH 3, at 4 C for 1 h. After lyophylization the samples were reconstituted for measurement of cAMP by RIA (23).

For estimation of cellular protein content, cells in 24-well plates were washed with PBS and solubilized with 0.5% SDS. The SDS fractions were kept at -20 C until protein determination with the DC-protein assay (Bio-Rad Laboratories, Hercules, CA) using BSA as standard.

Data analysis
IC50 and EC50 values were calculated by nonlinear regression analysis, and specific binding data of saturation binding experiments were analyzed using the equation for binding to one binding site using Fig.P 6.0 (Biosoft, Cambridge, UK). Data are expressed as means ± SEM. The significance of difference between means was calculated by ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Retinoic acid induced differentiation of P19 cells
Differentiation into neurons and glial cells of aggregated P19 cells was obtained through treatment with 1 µM RA for 4 days (19, 20). Neurons and astrocytes, identified with specific monoclonal antibodies to NF160 or GFAP, were not observed in untreated P19 cells (Fig. 1Go, A–C). Neurons were initially visualized at about day 6 of culture after the start of the RA treatment. On day 10, a higher density of NF160 positive neurons was observed (Fig. 1Go, D and E); less than 1% of the cells being then stained for GFAP (Fig. 1FGo). At 14 days neurons were only exceptionally visualized while at day 22 the density of GFAP-positive astrocytes were increased to over 90% (Fig. 1Go, G and I). At this time neurons were not observed (Fig. 1Go, G and H).



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  		Figure 1. Characterization of neurons and astrocytes derived from RA-treated P19 cells by immunohistochemistry. Untreated P19 cells (A–C; 2 days of culture) and cells treated with 1 µM RA on days 10 (D–F) and 24 (G–J) after the start of the 4 days RA treatment period. The cells were stained with specific antibodies to NF160 (B, E, H) and GFAP (C, F, J) followed by DTAF-labeled anti-IgG. Phase contrast (A, D, G) and corresponding immunofluorescence (B, E, J). C, F, and H are cultures grown in parallel and stained with the respective antibodies. Bar, 50 µm.

 
When P19 cells were treated with RA while attached to the surface of the culture dishes nonidentified presumably fibroblast-like cells were induced (18, 19). The above used antibodies to NF160 and GFAP did not stain any of these fibroblast-like cells (not shown).

Receptor autoradiography
Before RA treatment all the P19 cells were labeled with [125I]sCT(1–32) (Fig. 2Go, A and B). 10 days of culture after the start of the RA treatment of the P19 cells, dense labeling was observed on flat cell types growing underneath neuron-like cells (Fig. 2Go, C and D). [125I]sCT(1–32) binding was never associated with morphologically identified neurons. [125I]sCT(1–32) labeling was not visible in cultured astrocytes on day 24 (Fig. 2Go, E and F). The characteristic grainy pattern observed in Fig. 2Go, B and D, was not seen in the presence of 1 µM sCT(1–32) (not shown).



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  		Figure 2. Autoradiography with [125I]sCT(1–32) of P19 cells before and after treatment with 1 µM RA. Untreated P19 cells (A, B; 2 days of culture) and P19 cells differentiated into neurons (day 10; C, D) and astrocytes (day 24; E, F) after the start of the 4 days RA treatment period were incubated with 1.25 nM [125I]sCT(1–32) and autoradiography was performed as described in Materials and Methods. A, C, and E are phase contrast images of the corresponding dark-field autoradiographs B, D, and F. Bar, 200 µm.

 
Expression of CT and PTH/PTHrP receptors in untreated P19 cells
Specific binding of 0.125 nM [125I]sCT(1–32) to untreated P19 cells reached an apparent steady-state at 15 C after 2 h, and was 56.3 ± 3.3 fmol/mg protein (n = 13) (Fig 3AGo). Nonspecific binding was less than 5% of specific binding. Binding of [125I]sCT(1–32) was essentially irreversible as previously reported (1) (not shown). Half-maximal binding of [125I]sCT(1–32) was obtained with 0.32 ± 0.07 nM (n = 3) with maximal binding of 350 ± 27 fmol/mg protein, corresponding to a receptor density of 100,000 ± 14,600 binding sites per cell (n = 3). Half-maximal binding inhibition of 0.125 nM [125I]sCT(1–32) by sCT(1–32) occurred with an IC50 of 2.3 ± 0.3 nM (n = 7) (Fig. 4AGo). The IC50 of the related peptides hCT, human calcitonin gene-related peptide-I (hCGRP-I) and human amylin were higher than 1 µM (not shown). Maximal cAMP accumulation by sCT(1–32) was 47 ± 5-fold over basal levels (12.7 ± 1.7 pmol/mg protein; n = 11) with an EC50 of 0.05 ± 0.01 nM (n = 7) (Fig. 5AGo). With hCT, human amylin and hCGRP-I the EC50 were 60-, 80-, and 220-fold higher (not shown).



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  		Figure 3. Time course of specific binding of [125I]sCT(1–32) and [125I]chPTHrP(1–36) and corresponding cAMP accumulation in P19 cells differentiating into neurons and astrocytes. Specific binding of 0.125 nM [125I]sCT(1–32) (A) and 0.125 nM [125I]chPTHrP(1–36) (B) and stimulation of cAMP by 100 nM sCT(1–32) (C) and 100 nM chPTHrP(1–36) (D) in P19 cells treated with 1 µM RA for 4 days, compared with untreated confluent control cells (U). Days refer to time in culture after the start of the 4 days RA treatment period. Values are means ± SEM of at least five independent experiments.

 


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  		Figure 4. Competition of [125I]sCT(1–32) and [125I]chPTHrP(1–36) binding during differentiation of P19 cells into neurons and astrocytes. Specific [125I]sCT(1–32) (A) and [125I]chPTHrP(1–36) binding (B) was displaced by sCT(1–32) and chPTHrP(1–36) in untreated confluent P19 cells ({square}) and after treatment of P19 cells with 1 µM RA on days 10 ({triangleup}) and 24 ({circ}) after the start of the 4 days RA treatment period. Values are means ± SEM of at least three independent experiments.

 


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  		Figure 5. Cellular cAMP accumulation evoked by sCT(1–32), chPTHrP(1–36) and rPTH(1–34) during development of P19-cells into neurons and astrocytes. cAMP was stimulated by sCT(1–32) ({square}), chPTHrP(1–36) ({triangleup}) and rPTH(1–34) ({circ}) in untreated confluent P19 cells (A) and on days 10 (B), 18 (C), and 24 (D) after the start of the 4 days RA treatment period. Values are means ± SEM of at least six independent experiments.

 
In untreated P19 cells specific binding of 0.125 nM [125I]chPTHrP(1–36) was undetectable (<0.5 fmol/mg protein) (Fig. 3BGo). But chPTHrP(1–36) and rPTH(1–34) maximally stimulated cAMP accumulation 3.4 ± 0.3-fold (n = 11) and 3.8 ± 0.8-fold (n = 4) over basal levels, with EC50 of 1.3 ± 0.6 nM (n = 4) and 2.0 ± 0.8 nM (n = 4), respectively (Fig. 5AGo).

Expression of CT and PTH/PTHrP receptors in cells differentiated into neural phenotypes
Specific binding of 0.125 nM [125I]sCT(1–32) was reduced to 16.5 ± 1.7 fmol/mg protein (n = 19) 10 days after the start of the RA treatment (day 10), at the peak of neuron appearance, and decreased further to 2.4 ± 0.3 fmol/mg protein (n = 12) on day 24 when mainly astrocytes were identified (Fig. 3AGo). Basal cAMP levels were 2-fold higher than in untreated control cells, and in the range of 17–27 pmol/mg protein. cAMP accumulation in response to 100 nM sCT(1–32) was comparable on day 10 (51 ± 4-fold basal; n = 19) to that in untreated cells. sCT evoked cAMP accumulation gradually decreased during development of astrocytes (day 24: 19 ± 2-fold-basal; n = 13; P < 0.05 compared with day 10) (Fig. 3CGo).

During neural differentiation specific binding of 0.125 nM [125I]chPTHrP(1–36) was higher than in untreated cells (Fig. 3BGo). Binding was similar at the peak of neuronal (2.8 ± 0.3 fmol/mg protein; n = 21) and astrocyte (3.1 ± 0.2 fmol/mg protein; n = 15) development. In parallel, maximal cAMP accumulation in response to 100 nM chPTHrP(1–36) was similarly raised when neurons were observed [day 10: 35 ± 4-fold-basal (n = 23)] and at maximal density of astrocytes (day 24: 37 ± 4-fold-basal (n = 16) (Fig. 3DGo).

Half-maximal binding inhibition of [125I]sCT(1–32) by sCT(1–32) during neural differentiation was comparable with that in untreated cells with IC50 values of 1.3 ± 0.5 nM (n = 10) on day 10 and 2.7 ± 1.8 nM (n = 3) on day 24 (Fig. 4AGo). The EC50 of sCT(1–32) stimulated cAMP accumulation on days 10, 18, and 24 of 0.37 ± 0.11 nM (n = 12), 0.61 ± 0.12 nM (n = 9) and 1.06 ± 0.23 nM (n = 8) were 7-fold (P < 0.001), 12-fold (P < 0.001) and 21-fold (P < 0.001) higher, respectively, than in untreated P19 cells (Fig. 5Go).

Half-maximal inhibition of [125I]chPTHrP(1–36) binding by chPTHrP(1–36) remained unchanged during neural differentiation (Fig. 4BGo). IC50 values were 1.0 ± 0.11 nM (n = 12) on day 10 and 1.6 ± 0.3 nM (n = 7) on day 24. EC50 values of cAMP accumulation in response to chPTHrP(1–36) were 1.8 ± 0.3 nM (day 10; n = 18) and 1.8 ± 0.4 nM (day 24; n = 11) (Fig. 5Go). Similar results were obtained with rPTH(1–34) with EC50 values of 3.2 ± 1.0 nM (day 10; n = 5) and 4.3 ± 1.6 nM (day 24; n = 5).

Expression of CT and PTH/PTHrP receptors in cells differentiated into mesodermal phenotypes
P19 cells attached to tissue culture dishes and treated with RA go into mesodermal differentiation (18). Specific binding of 0.125 nM [125I]sCT(1–32) was decreased much like in cells led into neural differentiation when treatet with RA in cell aggregates but was delayed (Fig. 6AGo). Specific [125I]sCT(1–32) binding remained higher between days 22 and 30 of culture after the start of the RA treatment (P < 0.001). Basal cAMP levels were about 2- to 3-fold higher than in untreated control cells, and in the range of 23–45 pmol/mg protein. Between days 10 and 12, maximal cAMP accumulation by sCT(1–32) was comparable in mesodermal and neuronal cells (Fig. 6CGo). But, on days 22–30 maximal stimulation remained unchanged in fibroblast-like cells and was higher than in cultured astrocytes (days 22–30) (P < 0.005).



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  		Figure 6. Specific binding of [125I]sCT(1–32) and [125I]chPTHrP(1–36) and corresponding stimulation of cAMP accumulation in P19 cells treated with 1 µM RA during aggregation as compared with P19-cells treated with 1 µM RA while attached to the surface of tissue culture dishes. Specific binding of 0.125 nM [125I]sCT(1–32) (A) and 0.125 nM [125I]chPTHrP(1–36) (B) and cAMP accumulation with 100 nM sCT(1–32) (C) and 100 nM chPTHrP(1–36) (D) in cells differentiated into neurons and astrocytes (black bars) and fibroblast-like cells (hatched bars), compared with untreated confluent P19 cells (white bars), on days 10–12 and 22–30 after the start of the 4 days RA treatment period. Values are means ± SEM of at least four independent experiments.

 
Specific binding of 0.125 nM [125I]chPTHrP(1–36) was similar in fibroblast-like and neural cells (Fig. 6BGo). Maximal cAMP accumulation in response to chPTHrP(1–36) was raised compared with untreated P19 cells (P < 0.005), but the stimulation was smaller than in neural cells (Fig. 6DGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There is evidence for the existence in the brain of CT- and PTH-like peptides and PTHrP, and of several isotypes of CT, PTH, and PTHrP receptors (4, 11, 12, 13, 14, 15, 16, 17, 24). CT and PTH stimulated cAMP production have been revealed in rat brain cells in primary culture (3). PTH, moreover, increased cAMP in cultured rat astrocytes and cytosolic free calcium in neurons (7, 8, 9). PTHrP is involved in the regulation of embryonic endochondral bone formation, hair follicle development, and branching morphogenesis during mammary gland development, suggesting a role of PTHrP as a differentiation factor (25, 26, 27, 28). In the present report, mouse embryonic carcinoma P19 cells, which are induced by RA to sequentially differentiate into neurons and astrocytes have been used to investigate the expression of CT and PTH/PTHrP receptors during neural development (18, 19, 20). Alternatively, by changing the RA treatment protocol, the cells were led into mesodermal differentiation with the appearance of fibroblast-like cells.

Receptor autoradiography with [125I]sCT revealed labeling of all the P19 cells before RA treatment. Subsequently, sCT binding was restricted to flat undifferentiated cells that gradually disappeared over time but was not associated with morphologically identified neurons and astrocytes. As a consequence, specific [125I]sCT binding and maximal sCT stimulated cAMP production continually decreased during cultivation. Labeling was still high in these cells, which may indicate that CT receptor density was not greatly affected, and therefore autoradiographically still detectable. The affinity of the CT receptor remained unchanged after RA treatment of cells, but the potency of sCT to stimulate cAMP accumulation was lowered presumably because of reduced coupling to G protein-activated adenylyl cyclase or down-regulation of adenylyl cyclase.

Specific [125I]chPTHrP binding was not detected in undifferentiated P19 cells but became recognizable in RA treated cultures of P19 aggregates coincident with the appearance of neurons and later of astrocytes. Increased [125I]chPTHrP binding was paralleled by 10-fold higher maximal cAMP accumulation evoked by chPTHrP s compared with untreated cells. The results are consistent with the induction of a PTH/PTHrP receptor before or at the onset of neuronal differentiation. A relatively low density of PTH/PTHrP receptors and/or a homogenous distribution in the cells committed to differentiation and in differentiated cells may explain nonrecognizable [125I]chPTHrP binding to neurons and/or astrocytes on autoradiographic examination.

The pattern of CT receptor disappearance with RA treatment and induction of PTH/PTHrP receptors was qualitatively the same during neuronal differentiation of aggregated P19 cells and with the generation of mesodermal cell types from attached cells. A similar response was observed in mouse F9 embryonic carcinoma cells differentiated into parietal endoderm by RA (29, 30). Immortalized cells from embryonic rat calvaria acquired osteoblastic characteristics upon treatment with RA, associated with induction of PTH-activated adenylyl cyclase (31). Similarly, treatment with RA or bone morphogenetic protein-2 of nonosteogenic mouse pluripotent cells induced osteoblast-like features together with increased cAMP responsiveness to PTH (32). In the more mature UMR106–06 osteoblast-like osteosarcoma cells, on the other hand, treatment with RA lowered PTH/PTHrP receptor expression, together with a lower potency of PTH stimulated cAMP production in the face of unaltered CT receptors (33).

In conclusion, neural as well as mesodermal differentiation of the pluripotent embryonic carcinoma P19 cells by RA is paralleled by suppressed CT receptors localized on embryonic cells. During the sequential appearance of neurons and astrocytes or fibroblast-like cells PTH/PTHrP receptors are induced, consistent with their expression in differentiated cells. The loss of CT receptors and induction of PTH/PTHrP receptors together with characteristic cytological and immunohistochemical findings reveals the relevance of cell differentiation markers.


   Footnotes
 
1 This work was supported by the Swiss National Science Foundation (Grant 31–43094.95) and the Kanton of Zurich. Back

Received August 27, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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