Expression of a parathyroid hormone-like protein and its receptor during differentiation of embryonal carcinoma cells.

Expression of a Parathyroid Hormone-Like Protein and Its Receptor during Differentiation of Embryonal Carcinoma Cells

Samuel D. H. Chan, Gordon J. Strewler, Kathleen L. King, and Robert A. Nissenson Endocrine Unit Veterans Administration Medical Center and the Departments of Medicine and Physiology University of California San Francisco, California 94121

INTRODUCTION

Differentiation of mouse embryonal carcinoma cells to the parietal endoderm phenotype is associated with expression of PTH-responsive adenylate cyclase. A PTH-like protein (PLP), which binds to PTH receptors and activates adenylate cyclase in classical PTH target cells was recently isolated and cloned. We assessed whether the parietal endoderm phenotype is associated with the expression of PLP or its receptor. A 1.4-kilobase PLP transcript was detected in the mouse parietal endoderm cell line PYS-2. No hybridizing transcripts were evident in undifferentiated mouse embryonal carcinoma cells PSA-1 or F9. However, differentiation of these cells to parietal endoderm, either spontaneously (PSA-1) or by treatment with retinoic acid and dibutyryl cAMP (F9), resulted in expression of the 1.4-kilobase PLP message. Undifferentiated F9 cells displayed negligible specific binding of [12Sl]PLP-(1-34)amide. When F9 cells were induced to differentiate to parietal endoderm, specific binding sites for [125I]PLP(1-34)amide were expressed in parallel with PLPresponsive adenylate cyclase. These receptors, like those in classical PTH target tissues, displayed identical affinity (Kd = 5.2 niu) for bPTH-(1-34) and hPLP(1-34)amide; with binding capacity (Bmax) of 6.6 x 104 sites/cell. In the presence of retinoic acid, exogenous PLP substituted for dibutyryl cAMP in a concentration-dependent fashion in promoting the differentiation of F9 cells to parietal endoderm. Thus, both PLP mRNA and PLP receptors coupled to adenylate cyclase are expressed during the differentiation of mouse embryonal carcinoma cells. Increased cAMP levels produced by autocrine stimulation of PLP receptors by PLP may contribute to differentiation of embryonal carcinoma cells into parietal endoderm. (Molecular Endocrinology 4: 638646,1990)

Mouse embryonal carcinoma cells, the undifferentiated stem cells of teratocarcinoma, have many properties in common with pluripotent embryonic cells and, thus, provide a useful in vitro model system for exploring biochemical events associated with normal embryonic development (1, 2). One such cell line (F9), derived from a testicular teratocarcinoma of strain 129 mouse, can be either maintained as a stable population of undifferentiated stem cells under normal culture conditions (3) or induced to differentiate into extraembryonic endoderm cells (4). When treated with retinoic acid, adherent F9 cells differentiate into parietal endoderm, secreting large quantities of extracellular matrix proteins, such as type IV collagen and laminin, and expressing plasminogen activator (5). The differentiation process is enhanced by dibutyryl cAMP, isobutylmethylxanthine, or cholera toxin, all of which raise cellular cAMP levels (6-8). These results implicate cAMP as an important determinant of differentiation in mouse embryonal carcinoma cells and potentially in normal embryonic cells. However, little is known concerning the factors that regulate cAMP production in such cells. Embryonal carcinoma cell lines with both constitutive (PYS-2, a mouse parietal yolk sac embryonal carcinoma cell line) and induced (F9) parietal endoderm phenotypes are known to express a PTH-responsive adenylate cyclase activity (9, 10). It has yet to be determined whether the activation of this pathway contributes to the differentiation and/or function of parietal endoderm cells. It is also uncertain whether expression of PTH or other ligands capable of activating this pathway occurs during early mouse embryogenesis. Recently, a tumor-derived protein with a spectrum of biological activities similar to that of PTH has been purified, and its amino acid deduced from cloned cDNA (11-14). This 141-amino acid PTH-like protein (PLP) has 8 of the amino-terminal 13 residues identical to

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PLP and Embryonal Carcinoma Cell Differentiation

those present in human PTH (84-amino acid protein), but shows little similarity to other regions of PTH thought to be important for binding of the protein to receptors. Nevertheless, PLP and its biologically active, synthetic 1-34 amino-terminal fragment bind with high affinity to adenylate cyclase-coupled PTH receptors in bone and kidney. A number of normal tissues and cells have also been found to express this protein, but its function remains unclear (14-18). PTH-responsive adenylate cyclase in differentiated embryonal carcinoma cells might constitute a response system by which PLP regulates the differentiation and/ or function of parietal endoderm. As a first step toward testing this hypothesis, we assessed the expression and actions of PLP during embryonal carcinoma cell differentiation. The results demonstrate that PLP mRNA and PLP receptors coupled to adenylate cyclase are expressed during embryonal carcinoma cell differentiation. Furthermore, exogenous PLP enhances retinoic acid-induced differentiation of F9 cells to parietal endoderm. An autocrine pathway for induction or maintenance of the parietal endoderm phenotype by PLP is suggested.

RESULTS Stimulation of F9 Cell Differentiation by PLP Expression of laminin B1 and ^(IVJ-collagen message were used as biochemical markers to monitor the differentiation of F9 stem cells into parietal endoderm cells in monolayer cultures. Neither the 6.4-kilobase (kb) cv^lVJ-collagen nor the 6.2-kb laminin B1 mRNAs could be detected in undifferentiated F9 cells (Fig. 1). Treatment of F9 cells for 5 days with retinoic acid (1 MM) resulted in the expression of both a^lVJ-collagen and laminin B1 transcripts. Expression of mRNA for the two differentiation-specific proteins was further enhanced when the cells were exposed to retinoic acid (1 MM) together with either dibutyryl cAMP (1 ITIM) or hPLP-(1 34) amide (20 nM). In the presence of retinoic acid (1 MM), hPLP-(1-34)amide (20 nM) and dibutyryl cAMP (1 mM) provoked 3- to 5-fold and a 2- to 3-fold increases, respectively, in the expression of ^(IVJ-collagen and laminin B1 messages compared to F9 cells treated with retinoic acid alone (determined by densitometry). Similarly, bPTH-(1-34) (20 nM) plus retinoic acid (1 MM) produced a 3- to 4-fold increase in the level of ai(IV)collagen mRNA over retinoic acid (1 MM) treatment alone. Thus, in the presence of retinoic acid, both PLP and PTH were effective in promoting the differentiation of F9 cells to parietal endoderm. To confirm that increases in endogenous cAMP enhance differentiation, F9 cells were cultured for 5 days with retinoic acid (1 MM) and the adenylate cyclase activator forskolin (5 MM). Northern blot analysis of RNA isolated from these cells revealed that forskolin enhanced differentiation to an extent comparable to that produced by bPTH-(1-34) (20 nM; Fig. 1).

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Fig. 1. Expression of Laminin B1 mRNA and a^l mRNA in Differentiated F9 Cells Total RNA was used for hybridization to 32P-labeled probes for a^lVJ-collagen (A) and laminin B1 (B). The lanes contain 10 ^g RNA from control F9 (lane 1) or F9 cells treated for 5 days with 2) retinoic acid (1 MM). 3) retinoic acid (1 MM) and hPLP(1 -34)amide (20 nM), 4) retinoic acid (1 HM) and dibutyryl cAMP (1 mM), 5) retinoic acid (1 HM) and bPTH-(1-34) (20 nM), and 6) retinoic acid (1 HM) and forskolin (5 fiM). The positions of the 28S and 18S ribosomal RNAs and the estimated sizes of ^(IVJ-collagen (6.4 kb) and laminin B1 (6.2 kb) mRNAs are indicated.

The effect of PLP on differentiation of F9 cells in the presence of retinoic acid was concentration dependent (Fig. 2). In the absence of retinoic acid, PLP was ineffective. Expression of Adenylate Cyclase-Coupled PLP Receptors in Embryonal Carcinoma Cells with Parietal Endoderm Phenotypes The expression of PLP receptors in mouse embryonal carcinoma cells was assessed by [125I]PLP-(1 -34)amide binding (Fig. 3). The specific PLP binding of undifferentiated F9 cells was approximately 2.5%. Retinoic acid treatment (1 MM; 5 days) resulted in a 4-fold increase in [125l]PLP-(1-34)amide binding. Expression of PLP receptors was further enhanced 6- to 7-fold when F9 cells were cultured in the presence of retinoic acid (1 MM) and dibutyryl cAMP (1 mM). In the presence of retinoic acid (1 MM), hPLP-(1-34)amide (20 nM) could substitute for dibutyryl cAMP in triggering increased PLP receptor expression. Specific PLP binding in PYS2 cells was similar to that in differentiated F9 cells. The time course of expression of adenylate cyclasecoupled PLP receptors in differentiating F9 cells was assessed. F9 cells were induced to differentiate by treatment with retinoic acid and dibutyryl cAMP for 1 5 days. Significant expression of PLP receptors and PLP-responsive adenylate cyclase occurred after 2-3 days of treatment (Fig. 4). There was a steep rise in


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- 6 . 2 Kb Fig. 2. Effects of hPLP-(1 -34)amide on ^(IVJ-Collagen mRNA and Laminin B1 mRNA Expression in F9 Cells F9 cells were treated for 5 days with 0.2 nM hPLP-(1 -34) amide (lane 2), 2 nM hPLP-(1 -34)amide (lane 3), 20 nM hPLP(1 -34)amide (lane 4), 1 ^ M retinoic acid (lane 5), 1 HM retinoic acid and 0.2 nM hPLP (1 -34)amide (lane 6), 1 MM retinoic acid and 2 nM hPLP-(1-34)amide (lane 7), and 1 MM retinoic acid and 20 nM hPLP-(1-34) amide (lane 8). Lane 1 represents control F9 cells. Ten micrograms of total RNA were subjected to Northern analysis. A, Alphai(IV)-collagen message; B, laminin B1 message. The estimated sizes of mRNAs in kilobases are indicated.

specific [125l]PLP-(1-34)amide binding after 2 days of treatment (Fig. 4A), which appeared to parallel the rise in adenylate cyclase activity, as assessed by the response of F9 cells to hPLP-(1-34)amide, bPTH-(1-34), and forskolin (Fig. 4B). Both hPLP-(1-34)amide and bPTH-(1-34) exhibited similar stimulatory effects on adenylate cyclase activity. In F9 cells with the induced parietal endoderm phenotype, PLP-binding sites were identified using [125I] PLP-(1-34)amide(Fig. 5). Human PLP-(1-34)amideand bPTH-(1-34) bound to PLP receptors in differentiated F9 cells with equal potency. The unlabeled biologically active 1-34 peptides competed for these sites with an IC50of 5-6 nM. Scatchard analysis of the data indicated that PLP receptors in differentiated F9 cells displayed identical affinity (Kd = 5.2 nM) for hPLP-(1-34)amide and bPTH-(1-34), with a binding capacity (Bmax) of 6.6 x 104 sites/cell (Fig. 5, inset). PLP receptors in PYS-2 cells displayed a similar affinity for hPLP-(1 -34)amide and bPTH-(1-34) (data not shown). Expression of PLP mRNA in Embryonal Carcinoma Cells with Parietal Endoderm Phenotypes We evaluated expression of PLP mRNA in three mouse embryonal carcinoma cell lines (Fig. 6A). PYS-2 cells, which display a stable parietal endoderm phenotype,

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Fig. 3. Expression of PLP Receptors in Embryonal Carcinoma Cells Associated with Parietal Endoderm Phenotype F9 cells were treated for 5 days with 1 HM retinoic acid (RA), 1 HM retinoic acid and 20 nM hPLP-(1 -34)amide (RA+PLP), or 1 nM retinoic acid and 1 mM dibutyryl cAMP (RA+diBcAMP). Specific [125l]PLP-(1-34)amide binding is the difference between binding in the absence and presence of 20 nM bPTH(1 -34). Each point is the mean ± SE of three to five experiments, each performed in triplicate. PYS represents PYS-2 cells.

expressed a 1.4-kb PLP transcript, whereas 786-0 human renal carcinoma, as previously reported (13), expressed multiple transcripts of 2.2, 1.8, and 1.5 kb. Undifferentiated F9 and PSA-1 mouse embryonal carcinoma stem cells did not express detectable PLP mRNA. However, differentiation to the parietal endoderm phenotype, whether spontaneous (PSA-1) or induced by retinoic acid and dibutyryl cAMP (F9), was associated with the expression of a 1.4-kb PLP mRNA transcript. Comparable levels of PLP mRNA were detected in F9 cells treated with either 0.1 or 1 HM retinoic acid and in the absence or presence of 1 mM dibutyryl cAMP (Fig. 6B). The level of PLP mRNA expression in parietal endoderm cells was found to be very low, as autoradiography had to be carried out for 2-3 days before the 1.4-kb signal could be visualized. Time-Course Correlation between PLP mRNA Expression and F9 Cell Differentiation F9 cells were cultured in monolayers with retinoic acid (1 (IM) for 1-5 days, and the expression of «i(IV)collagen and PLP mRNAs was determined by Northern analysis. The result indicated that expression of PLP mRNA precedes the retinoic acid-induced differentiation of F9 cells. The 1.4-kb PLP message was detected after 1 day of retinoic acid treatment (Fig. 7B). The level of expression of the message appeared to remain more or less constant throughout the time course of retinoic acid treatment. On the other hand, 1 day of retinoic acid treatment did not induce any discernable differen-


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0 1 2 3 4 5 Days of Exposure to RA* diBcAMP Fig. 4. Time Course for Expression of Adenylate CyclaseCoupled PLP Receptor in F9 Cells F9 cells were exposed to retinoic acid (1 /XM) and dibutyryl cAMP (1 ITIM; RA+diBcAMP) for 1-5 days before assessment of specific [1Z5l]PLP-(1-34)amide binding (A) or adenylate cyclase activity (B). Values are the mean ± SE of four experiments, each performed in triplicate.

tiation of F9 cells, as indicated by the inability to detect any cv^lV^collagen mRNA, the biochemical marker for parietal endoderm (Fig. 7A). Expression of a^lVJ-collagen mRNA was evident after 2 days of exposure to retinoic acid and increased further with prolonged treatment with retinoic acid.

DISCUSSION

PLP, a protein that was first isolated from human tumors associated with humoral hypercalcemia of malignancy (19-23), appears to elicit its PTH-like effects by interacting with PTH receptors in bone and kidney (2428). Recently, PLP was also found to be present in milk (29); in a number of normal adult and fetal tissues such

as lactating mammary gland, adrenal glands, pancreas, brain, and keratinocytes (14-17); as well as in chicken embryo (18), although its physiological functions remain obscure. In this study we demonstrate that differentiation of embryonal carcinoma stem cells to parietal endoderm is accompanied by expression of PLP mRNA and PLP receptor-coupled adenylate cyclase. Furthermore, in the presence of retinoic acid, PLP promotes the differentiation of F9 cells to parietal endoderm. We propose that PLP may play a role in promoting the biochemical events involved in normal embryonic development and differentiation through the generation of cAMP. Alterations in hormonal responsiveness of F9 embryonal carcinoma cells associated with differentiation was demonstrated in our study. In undifferentiated F9 stem cells, which express negligible levels of PLP receptors, the adenylate cyclase system is relatively insensitive to PLP, PTH, or forskolin. Differentiation of F9 cells toward parietal endoderm is characterized by a progressive increase in stimulation of cAMP production in response to PLP, PTH, or forskolin, with a concomitant expression of PLP receptors. Significant expression of PLP receptors and PLP-responsive adenylate cyclase occurs after 2-3 days of exposure of F9 cells to retinoic acid and dibutyryl cAMP and reaches maximal values by the fifth day. This time course parallels the appearance of morphological and biochemical markers of F9 cell differentiation, such as laminin, type IV collagen, and plasminogen activator (5-8). Similar changes in the PTH responsiveness of F9 cells were first observed by Liapi et al. (10) and Evain et al. (9). They reported that undifferentiated F9 stem cells respond to calcitonin with an increase in cAMP production. Differentiation of F9 cells to parietal endoderm causes a loss in the calcitonin responsiveness of the adenylate cyclase system. The differentiated F9 cells, on the other hand, were reported to possess a PTHresponsive adenylate cyclase system. Waterman et al. (30) also noted the acquisition of an adenylate cyclase highly responsive to PTH during prenatal development of the maxillary palatal complex in the golden hamster. We have previously shown that synthetic PLP amides bind to adenylate cyclase-coupled PTH receptors in canine renal plasma membranes and rat osteoblast-like osteosarcoma cells (UMR-106) with an affinity identical to that of bPTH-(1-34) (25). Moreover, photoaffinity cross-linking experiments revealed that PLP and PLP peptides both interact with an 85-kDa receptor in canine renal plasma membrane (25, 28). Similar observations have been reported in ROS 17/2.8 clonal rat osteosarcoma cells (26, 27). In the present study we observed that receptors in differentiated embryonal carcinoma cells display similar affinity (Kd = 5.2 nwi) for bPTH-(134) and hPLP-(1-34)amide, and that both of the 1-34 biologically active peptides were able to provoke similar activation of adenylate cyclase activity in differentiated F9 cells. It is, thus, likely that the biological activities of PLP in embryonal carcinoma cells, like those in bone and kidney, are derived from its activation of adenylate


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Hormone, nM Fig. 5. Binding of [125l]PLP-(1-34)amide to Differentiated F9 Cells Competitive inhibition by hPLP-(1 -34)amide (•) and bPTH-(1-34) (O) of [125l]PLP-(1-34)amide binding in F9 cells treated for 5 days with 1 ^M retinoic acid and 1 mM dibutyryl cAMP (RA+diBcAMP) compared with that in control F9 cells. Values are the mean ± SE of two experiments, each performed in triplicate. Inset, Scatchard analysis of [125l]PLP-(1-34)amide binding to retinoic acidand dibutyryl cAMP-treated F9. B/F, Bound to free ratio.

cyclase coupled to a common PTH/PLP receptor. This is further supported by the present observation that both hPLP-(1-34)amide and bPTH-(1-34) can promote the differentiation of F9 cells in the presence of retinoic acid. Our findings that PLP, PTH, and forskolin can substitute for dibutyryl cAMP in promoting the differentiation of F9 cells strongly implicates endogenous cAMP as a mediator of the effect of PLP and PTH. In the absence of retinoic acid, however, PLP does not promote F9 cell differentiation. This is consistent with previous observations that retinoic acid is required for transition of embryonal carcinoma cells to a phenotype that is responsive to the differentiating effects of cAMP (7, 8). The expression of PLP mRNA is associated with the parietal endoderm phenotype in mouse embryonal carcinoma cells. We found that the message is present in spontaneously differentiated PSA-1 cells as well as in embryonal carcinoma cells with both constitutive (PYS2) and induced (F9) parietal endoderm phenotypes. In contrast to the heterogeneity of the human PLP transcript that arises from both alternative splicing of exons and use of multiple promoters (13, 31-33), differentiated mouse embryonal carcinoma cells express only a single 1.4-kb PLP mRNA. Others have also reported expression of a single major 1.3- to 1.5-kb PLP transcript in various rodent tissues and cell lines (14, 17, 34). It, thus, appears that the structure and organization of rodent PLP genes may be less complex then those of the human gene. We observed that PLP mRNA expression is an early

event in the differentiation of F9 cells when they were treated with retinoic acid. Moreover, when F9 cells were treated with either retinoic acid or retinoic acid and dibutyryl cAMP, they showed a similar level of PLP mRNA expression. Hence, PLP mRNA expression is triggered by retinoic acid alone and may be independent of cAMP, as might be expected if PLP were an endogenous trigger of differentiation. It should be noted, however, that we have been unable to detect immunoreactive PLP in conditioned medium from retinoic acid-treated F9 cells using a sensitive RIA (35). Furthermore, as described above, expression of PLP receptors in response to retinoic acid lags behind that of PLP itself and occurs with a time course similar to that of other markers of differentiation. Further studies are required to define the role, if any, of PLP as an autocrine or paracrine differentiation factor in embryonal carcinoma cells. Biochemical, morphological, and immunological studies have demonstrated similarities between mouse embryonal carcinoma stem cells and the inner cell mass (ICM) of the blastocyst (1, 2). We speculate that PLP may play an important role during ICM differentiation and embryo development. The mouse blastocyst consists of an outer monolayer of trophectoderm and an internal group of cells, the ICM. During subsequent development, cells in the ICM differentiate into two types of extraembryonic endoderm, the parietal endoderm and the visceral endoderm. One possible role of PLP may be to promote the differentiation of the ICM and migration of parietal endoderm cells along the trophectoderm layer as the yolk sac cavity enlarges



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Fig. 6. Expression of PLP mRNA in Differentiated F9 Cells A, A Northern blot of 5 ^g poly(A)+ RNA was hybridized with a 32P-labeled cDNA probe for human PLP (10B5) (13). Lane 1, Control F9; 2, F9 treated with 0.1 n** retinoic acid and 1 mM dibutyryl cAMP for 5 days; 3, undifferentiated PSA-1; 4, differentiated PSA-1 (Normally, PSA-1 cells can be maintained in the undifferentiated state by frequent subculture in conjunction with fibroblast feeder cells. When cultured in the absence of feeder cells, PSA-1 is stimulated to develop cell aggregates that differentiate, with resultant formation of a layer of parietal endoderm cells covering the entire outer surface of these aggregates.); 5, PYS-2. The Northern blot was kindly provided by Dr. Gail Martin (Department of Anatomy, University of California, San Francisco, CA). The blot was exposed to x-ray film for 48 h. B: Lane 1,10 ^g total RNA from human renal carcinoma cell line 786-0 was used as a positive control for PLP mRNA expression. Five micrograms of poly(A)+ RNA from PYS-2 (lane 2), undifferentiated F9 (lane 3), F9 treated with 0.1 HM retinoic acid for 5 days (lane 4), F9 treated with 1 HM retinoic acid for 5 days (lane 5), F9 treated with 0.1 H*A retinoic acid and 1 mM dibutyryl cAMP for 5 days (lane 6), and F9 treated with 1 HM retinoic acid and 1 mM dibutyryl cAMP for 5 days (lane 7). Autoradiography was carried out for 8 h for lane 1 and for 60 h for lanes 2-7. The positions of the 28S and 18S ribosomal RNAs and the estimated sizes of PLP mRNAs are indicated.

during gestation. The association of PLP receptors with the parietal endoderm phenotype also raises the possibility that PLP may play a role in regulating the synthesis and/or secretion of basement membrane components by parietal endoderm cells. Additional studies are warranted to define the expression and actions of PLP during mammalian embryogenesis.

MATERIALS AND METHODS Materials Synthetic bPTH-(1-34) was purchased from Bachem, Inc. (Torrance, CA). Human PLP-(1 -34)amide was a generous gift of Dr. Michael Rosenblatt (Merck, Sharp, and Dohme Research


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B -1.4kb 3 4 5 Days Fig. 7. Time Course for Retinoic Acid-Induced Expression of a,(IV)-Collagen and PLP mRNAs in F9 Cells Northern hybridization of RNA from F9 cells treated with retinoic acid (1 HM) for 1-5 days. A, Ten micrograms of total RNA hybridized to a 32P-labeled cDNA probe for ^(IVJ-collagen. B, 5 ng poly(A)+ RNA hybridized to a 32P-labeled human PLP cDNA probe. PYS, PYS-2. Lane 0, Untreated F9. 786-0, Five micrograms of total RNA from human renal carcinoma cell line 786-0 as a positive control for PLP expression. Autoradiography was carried for 20 h for 786-0 and 48 h for the rest of the lanes.

Laboratories, West Point, PA). Mouse embryonal carcinoma cell lines F9 and PYS-2 were kindly provided by Dr. Gail Martin (University of California, San Francisco, CA). cDNA probes for mouse laminin B1 (pPE47) and ^(IVJ-collagen (pPE131) were gifts of Dr. Brigid Hogan (Vanderbilt University, Nashville, TN). Cell Culture PYS-2 cells were grown at 37 C in Dulbecco's Modified Eagle's Medium (DME) containing 10% calf serum, antibiotics (50 U/ ml penicillin and 50 U/ml streptomycin), and 4 ITIM glutamine in a 5% CO2 humidified incubator. F9 cells were cultured in the same medium on gelatin-coated culture dishes. For the differentiation studies, F9 cells were treated for 5 days with retinoic acid (1 HM), retinoic acid (1 HM) plus dibutyryl cAMP (1 mM), or retinoic acid (1 HM) plus hPLP-(1 -34)amide (20 nM), or otherwise as stated in the figure legends. For the time-course study of differentiation, the cells were exposed to retinoic acid (1 HM) and dibutyryl cAMP (1 mM) for 1-5 days. All treatments were started 1 day after plating the cells, and the cells reached confluence by the sixth day. Retinoic acid was added as a concentrated stock in ethanol to the medium. The final concentration of ethanol in the medium (0.01 %) did not alter cell morphology, growth, or biochemical responsiveness. [12Sl]PLP-(1-34)Amide Binding Assay Human PLP-(1-34)amide was iodinated to a specific activity of 70-100 MCi/^g- as previously described (25). Initial experiments indicated that steady state binding of labeled ligand occurred after 20 min for PYS-2 and after 90 min for F9 at 20 C. These conditions were adopted in all subsequent experiments. Confluent PYS-2 or F9 cells grown in six-well cluster plates were preincubated in 0.7 ml DME supplemented with 20 mM HEPES for 60 min at 20 C. [125l]PLP-(1-34)amide, in the absence or presence of excess unlabeled bPTH-(1-34) or hPLP-(1-34)amide, was added. At the end of incubation, the cells were washed three times with 1.0 ml ice-cold PBS and were then solubilized with 1.0 ml 75 mM NaOH for 7-counting.

The adenylate cyclase activity of the F9 cells was measured in six-well cluster plates by the conversion of [3H]ATP to [3H] cAMP (36). F9 cells were preincubated with 1.0 ml serum-free DME for 1 h at 37 C. The ATP pool of the cells was then labeled by incubation with 1 nC\ [3H]adenine for 2 h at 37 C. After aspiration of the medium, the cells were washed twice with 1.0 ml calcium- and magnesium-free PBS, then incubated for 10 min with 0.4 M isobutylmethylxanthine in 1.0 ml serumfree DME. Bovine PTH-(1-34), hPLP-(1 -34)amide, orforskolin was added, and the incubation was continued for an additional 10 min at 37 C. The reaction was stopped by adding 0.5 ml 2% sodium dodecyl sulfate (SDS), followed by 0.5 ml 20% trichloroacetic acid. Carrier solution (0.1 ml) containing 5 mM each of adenine, adenosine, AMP, ADP, ATP, and cAMP was added. [14C]cAMP (3000 cpm) was included as a standard to assess recovery. The reaction mixture was frozen at —20 C, thawed, vortexed, and centrifuged at 1500 x g for 10 min. Cyclic AMP in the supernatant was then isolated by serial Dowex X-50 and alumina column chromatography (37). Radiolabeled cAMP was collected and counted by liquid scintillation. Each sample was corrected for the recovery of [14C] cAMP, which averaged 60%. RNA Isolation Total RNA was isolated by a modification of the method of Chomczynski and Sacchi (38). Confluent cells grown in 10-cm culture dishes were dispersed in 2.0 ml lysis solution [4 M guanidinium thiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% sarcosyl, and 0.1 M 2-mercaptoethanol], and the cells were removed from the culture dish by scraping. Sequentially, 0.2 ml 2 M sodium citrate (pH 4.0), 2 ml water-saturated phenol, and 1.0 ml chloroform-isoamyl alcohol (49:1, vol/vol) were added. The mixture was vortexed for 10 sec and cooled on ice for 15 min. After centrifugation at 10,000 x g for 20 min at 4 C, RNA in the upper aqueous phase was precipitated with 2 vol ethanol at - 2 0 C for 2 h. The RNA was pelleted by sedimentation at 10,000 x g for 20 min at 4 C and resuspended in 1.0 ml lysis solution. Phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation were repeated. The resulting RNA pellet was dissolved in 100 n\ DEPC-treated water. Poly(A)+ RNA was prepared by affinity chromatography on oligo(dT)-cellulose. Northern Analysis Total RNA or poly(A)+ RNA was fractionated on 1 % agaroseformaldehyde gel and transferred to Hybond-N nylon membrane (Amersham, Arlington Heights, IL). Blots were hybridized with 32P-labeled cDNA probe for mouse a^lVJ-collagen (pBE131) (39, 40), mouse laminin B1 (pBE47) (41), or human PLP (10B5) (13) in 0.2 M sodium phosphate (pH 7.2); 1 % BSA; 35% formamide; 1 mM EDTA; and 7% SDS for 16 h at 65 C. After washing in 1 x SSC-1% SDS at 65 C for the detection of PLP message or 1 x SSC-1% SDS followed by 0.1 x SSC0.1% SDS at 65 C for the detection of laminin B1 and ^(IV)collagen mRNAs, the blots were exposed to Kodak XAR-5 film at - 7 0 C with intensifying screens (Eastman Kodak, Rochester, NY). The sizes of the mRNAs were determined using molecular size standards (0.24- to 9.5-kb RNA ladder; BRL, Gaithersburg, MD).

Acknowledgments We thank Dr. Brigid Hogan for the a,(IV)-collagen (pBE131) and laminin B1 (pBE47) cDNA probes, and Dr. Gail Martin for embryonal carcinoma cell lines F9 and PYS-2 and the Northern blot. We are also grateful to Dr. Michael Rosenblatt for providing us with hPLP-(1 -34)amide.


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Received December 7,1989. Revision received January 26, 1990. Accepted January 26,1990. Address requests for reprints to: Dr. Samuel D. H. Chan, Endocrine Unit, Veterans Administration Medical Center (111N), 4150 Clement Street, San Francisco, California 94121. This work was supported by National Research Service Award DK-07418, NIH Grants CA-34738 and DK-35323, and the Research Service of the Department of Veterans Affairs.

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Differential expression of jun and fos genes during differentiation of mouse P19 embryonal carcinoma cells.

Expression of a 91-kilodalton PEA3-binding protein is down-regulated during differentiation of F9 embryonal carcinoma cells.

Neural differentiation increases expression of Alzheimer amyloid protein precursor gene in murine embryonal carcinoma cells.

Expression of transcripts of complement components and their receptors during differentiation of embryonal carcinoma cell lines.

Differentiation of embryonal carcinoma cells in response to retinoids.

Histone H1(0) mRNA and protein accumulate early during retinoic acid induced differentiation of synchronized embryonal carcinoma cells.

Differentiation as a requirement for simian virus 40 gene expression in F-9 embryonal carcinoma cells.

Ectopic expression of c-jun leads to differentiation of P19 embryonal carcinoma cells.

Chemically induced bidirectional differentiation of embryonal carcinoma cells in vitro.

Foxa1 contributes to the repression of Nanog expression by recruiting Grg3 during the differentiation of pluripotent P19 embryonal carcinoma cells.

E1a-dependent expression of adenovirus genes in OTF963 embryonal carcinoma cells: role of E1a-induced differentiation.

Differentiation and proliferation in mouse embryonal carcinoma cells.

Epidermal growth factor receptors increase during the differentiation of embryonal carcinoma cells.

Actin and myosin expression during development of cardiac muscle from cultured embryonal carcinoma cells.

C during in vitro-induced differentiation of F9 and P19 embryonal carcinoma cells.

Defective expression of adenovirus genes during early infection of undifferentiated OTF963 embryonal carcinoma cells.

Aggregation and cell cycle dependent retinoic acid receptor mRNA expression in P19 embryonal carcinoma cells.

Quantitative evaluation of anticalciuretic effects of synthetic parathyroid hormonelike peptides.

Determinants of retrovirus gene expression in embryonal carcinoma cells.

Differential expression of fetomodulin and tissue plasminogen activator to characterize parietal endoderm differentiation of F9 embryonal carcinoma cells.

Differential sorting of beta tubulin isotypes into colchicine-stable microtubules during neuronal and muscle differentiation of embryonal carcinoma cells.

D1 embryonal carcinoma cell differentiation.

8-Methyltryptanthrin-induced differentiation of P19CL6 embryonal carcinoma cells into spontaneously beating cardiomyocyte-like cells.

Differentiation of P19 embryonal carcinoma stem cells into insulin-producing cells promoted by pancreas-conditioned medium.