Molecular Vision 2000; 6:243-251 <http://www.molvis.org/molvis/v6/a33/> Received 2 December 1998 | Accepted 30 November 2000 | Published 8 December 2000

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Regulation of interphotoreceptor retinoid-binding protein (IRBP) gene expression by cAMP in differentiated retinoblastoma cells

Azza E. B. El-Remessy,^1,2 Ahmad M. Rabie,² Mamdouh M. El-Shishtawy,² Laila A. Eissa,² Gregory I. Liou¹
 
 

¹Department of Ophthalmology, Medical College of Georgia, Augusta, GA; ²Department of Biochemistry and Pharmacology, Faculty of Pharmacy, Mansura University, Mansura, Egypt

Correspondence to: Gregory I. Liou, Ph.D., Department of Ophthalmology, Medical College of Georgia, Augusta, GA, 30912; Phone: (706) 721-4599; FAX: (706) 721-7913; email: giliou@mail.mcg.edu

Abstract

Purpose: To determine the mechanism of cyclic AMP (cAMP) regulation of the interphotoreceptor retinoid-binding protein (IRBP) gene in retinoblastoma cells.

Methods: WERI-Rb1 cells pretreated with laminin or grown on poly-D-lysine-coated substratum for three days were treated with forskolin/3-isobutyl-1-methylxanthin (IBMX) or with dimethyl sulfoxide (DMSO). During a time course of 96 h, cell morphologies were determined by light microscopy, cellular cAMP levels measured by radioimmunoassay, and IRBP and b-actin gene expression determined by Northern blot and RNase protection analyses. IRBP expression and b-actin gene expression in these cells were also determined in the presence or absence of actinomycin D or cycloheximide.

Results: After laminin treatment for 3 days, 27-34% of WERI-Rb1 cells differentiated into spindle shapes. Further forskolin treatment in the presence of IBMX for 5 days resulted in many cells exhibiting the formation of long, ramifying, neurite-like processes that were abolished by an inhibitor of protein kinase A. Cells grown on poly-D-lysine-coated substratum treated with forskolin remained undifferentiated. Treatment of laminin-pretreated cells with forskolin/IBMX, but not with DMSO/IBMX, raised the cAMP level 15-fold within the first hour of treatment. Northern blot analysis of these cells showed a rapid increase of IRBP mRNA, but not b-actin mRNA, reaching a maximum of about 3-fold at 6-8 h. A similar increase of IRBP mRNA was observed using RNase protection analysis except that the maximum was observed at 2-4 h. Actinomycin D blocked this IRBP mRNA induction. Cycloheximide had no effect in this induction.

Conclusions: These results demonstrate that laminin induces WERI-Rb1 cell differentiation and cAMP provokes the formation of long ramifying neurite-like processes. Forskolin selectively induces IRBP gene expression in the laminin-treated cells through a cAMP-mediated pathway without de novo protein synthesis.

Introduction

In the mammalian visual system, normal development of the retina is essential for the establishment of a mature and functional multi-layered retinal structure capable of initiating vision and conducting phototransduction. The development of retina from embryonic neuroepithelium consisting of common retinal progenitor cells is believed to involve a coordinated process of mitotic division, cell fate determination, cell migration, and differentiation [1]. The molecular mechanisms underlying this developmental process remain largely unknown.

Cell differentiation is associated with cell type-specific gene expression. It is therefore critical to identify genes expressed in the retina during development and elucidate the regulatory mechanisms controlling their differential expression in the developing retina in order to understand the molecular basis governing retinal development. In photoreceptor cells, IRBP expression begins during the early stages of differentiation prior to outer segment formation [2]. IRBP is a retinoid- and fatty acid-binding glycoprotein found in the interphotoreceptor matrix between photoreceptor and retinal pigment epithelial cells, in pinealocytes, and in several retinoblastoma-derived cell lines (reviewed in [3]). The importance of IRBP for retinal development has been demonstrated by the recent generation of mice with a targeted disruption of the IRBP gene, which demonstrated early-onset photoreceptor abnormalities [4].

During terminal differentiation, tissue-specific gene expression is largely regulated at the transcriptional level. Studies of transcriptional regulation of photoreceptor-specific genes have recently been conducted in immortal photoreceptor cell lines derived from retinoblastoma tumors [5]. Two retinoblastoma cell lines, Y-79 and WERI-Rb1, were used in these studies [6,7]. These cells have provided a valuable model for the study of photoreceptor differentiation and development in vitro [8]. This model may have a direct relevance to early developing retina where there is substantial cell migration and differentiation before final attachment in specific strata.

It was reported that transcription of the IRBP gene in Y-79 cells was modulated by dibutyryl cyclic AMP (dbcAMP) in cells pretreated with laminin [9]. Laminin, a large heterotrimeric glycoprotein of the extracellular matrix and a natural basement membrane substratum [10], is known to promote adhesion, migration, and differentiation of various cells in vitro [11,12]. Cyclic AMP is a ubiquitous second messenger involved in numerous signal transduction pathways. Genes that are responsive to cAMP could be modulated through an induction of mRNA synthesis [13] or an increase in mRNA stability [14]. Responses to cAMP through mRNA synthesis are typically rapid, usually in hours or minutes, whereas responses to cAMP mediated through increased mRNA stability are typically slow, usually in 12 h to 1-2 days [14]. In the previous reports on the modulation of IRBP gene expression, the effects of various cAMP analogues were monitored at 5 days after the treatment [9,15]. The mechanism of the modulation effect of dbcAMP was not tested in the presence of actinomycin D, an inhibitor of RNA polymerase. Therefore, it is not clear whether the observed effect was due to an induction of mRNA synthesis or due to an increase in mRNA stability. In addition, because IRBP gene expression in Y-79 cells is also modulated by butyrate [15], it is not clear whether the effect of dbcAMP was due to butyrate or cAMP. In the current study, the effect of forskolin, an adenylate cyclase activator, in the presence of IBMX, a phosphodiesterase inhibitor, on laminin-pretreated WERI-Rb1 cells was determined by the analysis of cell morphology, cAMP level, and IRBP gene expression at different times of treatment. The mechanism of cAMP-mediated gene modulation was determined in the presence or absence of actinomycin D and cycloheximide.

Methods

Cell culture

The human retinoblastoma WERI-Rb1 (passage number 146, ATCC, Manassas, VA) cell line was grown in suspension in RPMI-1640 with L-glutamine (GIBCO BRL, Grand Island, NY), supplemented with 10% heat inactivated fetal bovine serum (GIBCO BRL), penicillin (100 U/ml) and streptomycin (100 mg/ml), in 75 cm² tissue culture flasks (Corning, Corning, NY) at 37 °C in a moist atmosphere of 95% air-5% CO₂. Medium was changed every 2-3 days. For all experiments that involved different treatments, cells were cultured in a medium containing 0.5% fetal bovine serum.

Reagents

All reagents used were from Sigma (St. Louis, MO) unless otherwise mentioned. Laminin was used to culture retinoblastoma cells in monolayers at a concentration of 12.5 mg/ml. Forskolin in DMSO was used at 10 mM. IBMX in DMSO was used at a level of 0.5 mM or 1 mM, as indicated. The protein kinase A (PKA) inhibitor, H89, was used at a concentration of 10 mM. The final concentration of DMSO in all experiments was 0.08%. Actinomycin D was used at 5 mg/ml. Cycloheximide was used at 10 mg/ml. In all experiments, forskolin and IBMX were either simultaneously added to cells or IBMX was added 30 min prior to forskolin, as indicated.

Cell morphology

To determine the morphological differentiation of WERI-Rb1 cells in the presence of laminin and forskolin, cells of 1 x 10⁵/ml/well were seeded on 6 well culture plates coated with poly-D-lysine (5 mg/cm²) or cultured in the presence of laminin. After 3 days, culture media were replaced with new media containing forskolin and IBMX or their solvent. The PKA inhibitor, H89, was used in combination with forskolin and IBMX. Cell morphology in 30-40 randomly selected fields (20-30 cells/field) of each well in different study groups were examined and quantified in the next 9 days using Nikon (Garden City, NY) contrast optics.

Cyclic AMP determination

The cAMP levels in cells cultured in the above described conditions were determined using a radioimmunoassay kit (Amersham, Little Chalfont, England) during a time course of various treatments. The cAMP determination kit is based on the method of Frandsen and Krishna [16] which depends on a competition between cAMP in a sample or standard and a tracer (¹²⁵I-cAMP) for a fixed number of antibody binding sites. At each time interval, cells were treated with 1 mM IBMX for 30 min prior to extraction and cAMP determination. Protein concentration of cells in each group was determined using a commercial assay (Bio-Rad Protein Assay, Bio-Rad, Richmond, CA) based on the method of Bradford [17].

Northern blot analysis

Cells cultured under the above conditions were subjected to Northern blot analysis during a time course of various treatments. The total RNA from each sample was isolated using the selective binding properties of silica gel-based membrane with the microspin technique RNeasy column kit (Qiagen, Chatsworth, CA). Equal amounts (10 mg) of total RNA, determined by absorbance at 260 nm, were electrophoresed on a 1% agarose gel in formaldehyde. RNA was blotted to Zeta-Probe GT blotting membrane (Bio-Rad) by capillary transfer and hybridized with probes for the human IRBP gene [18] or human b-actin gene [19]. The IRBP probe is a 1.2 kb cDNA extending from +2160 to +3400 cloned in the EcoRI and BamHI sites of pBluescript II KS (Strategene, Lajolla, CA). The b-actin probe is a 0.6 kb cDNA extending from +22 to +614 cloned in the HindIII and BamHI sites of pBluescript II KS. Probes were labeled with [a-³²P]dCTP with a Klenow fragment of DNA polymerase I using random oligomers (Pharmacia, Piscataway, NJ). Blotting, hybridization, washing, and autoradiography were all performed according to the manufacturer's instructions. The intensity of the bands for the IRBP and b-actin genes were scanned by E-C Densitometer (E-C Apparatus Corporation, St. Petersburg, FL) and analyzed with an HP (Wilmington, DE) integrator.

RNase protection analysis

Cells of the above described conditions also were analyzed by RNase protection. The analysis has been described previously [20]. Briefly, antisense RNA probes for the human IRBP and b-actin genes were synthesized using T7 polymerase. The template for the IRBP gene spans from +2160 to +3400 cloned in the EcoRI and BamHI sites of pBluescript II KS. The IRBP probe protects a 281 bp fragment of the coding strand from +3120 to +3400. The template for the human b-actin gene spans from +22 to +614 cloned in the HindIII and BamHI sites of pBluescript II KS. The actin probe protects a 177 bp fragment of the coding strand from +438 to +614. Linearized templates were labeled with [a-³²P]UTP and purified as described previously [20]. Total RNA (3 mg) was hybridized with each probe present in excess (500-800 pg or 4.0 x 10⁵ cpm). Single-stranded RNA was removed with RNase, purified, and subjected to electrophoresis on a denaturing 5% polyacrylamide and urea gel. Gels were dried and exposed to x-ray film with an intensifying screen. The intensity of the bands was quantified by scanning with a densitometer as described above.

Statistical analysis

Calculations were performed with the aid of a computer equipped with appropriate software (Microstat, Advanced Graphics Software Inc., Sunnyvale, CA) designed for filling and processing data as well as for statistical analysis. The results of investigated parameters were compared with the control using student's t-test. For multiple comparisons, single ANOVA was done followed by Post-hoc test (Scheffe). A p value of <0.05 was considered significant.

Results

Effect of forskolin and laminin on the morphology of WERI-Rb1 cells

A morphological study was performed on WERI-Rb1 cells under culturing conditions with various treatments. Initially, cells were either grown as monolayers on poly-D-lysine-coated substratum or treated with laminin. Under either condition, cells became attached to the culture plates immediately. Cells grown on poly-D-lysine-coated substratum remained morphologically undifferentiated throughout the entire study period of 12 days (Figure 1A). In contrast, cells pretreated with laminin for 3 days and and treated with forskolin and IBMX for 24 h appeared in spindle shape with short processes (window in Figure 1B). Five days later, many cells exhibited formation of long ramifying, neurite-like processes (Figure 1B).

To determine whether the above observed cell differentiation was induced by laminin alone or in combination with treatment with forskolin/IBMX, cells pretreated with laminin for 3 days were treated with the solvent DMSO. Cells treated in this manner also exhibited morphological differentiation at 24 h (window in Figure 1C) or 5 days (Figure 1C) of solvent treatment, but did not exhibit formation of long ramifying neurite-like processes.

To determine whether the effect of forkolin/IBMX treatment on cell differentiation involves a protein kinase A pathway, cells were pretreated with laminin for three days and then treated with forskolin/IBMX in the presence or absence of the protein kinase A inhibitor, H89. The cells treated with H89 appeared in spindle shape with one or more processes. However, these cells did not exhibit the long, ramifying, neurite-like appearance throughout the entire study period (Figure 1D). In the above studies, the percentage of differentiated cells in the presence of laminin/forskolin/IBMX (L/F/I) was significantly (p<0.05) higher than the other group treated with laminin alone (LN) or with laminin/forskolin/IBMX/H89(L/F/I/H). Comparison of the last two groups (LN and LN/F/I/H) revealed no significant difference (p>0.05) in the percentage of morphologically differentiated cells (Figure 1F).

To determine whether forskolin/IBMX alone also could induce cell differentiation, cells grown on poly-D-lysine-coated substratum were treated with forskolin/IBMX. These cells remained morphologically undifferentiated throughout the entire study period (Figure 1E). These results suggest that cell differentiation is induced by laminin. Forskolin/IBMX treatment, which presumably elevates cAMP levels, provokes the formation of long ramifying neurite-like processes. That provoking effect may be mediated by a protein kinase A pathway.

Effect of forskolin and laminin on the level of cAMP in WERI-Rb1 cells

To determine the cAMP-inducing effect of forskolin/IBMX, the levels of cAMP in laminin-pretreated cells were determined during a time course of forskolin treatment between 0 and 48 h. For all time intervals of forskolin treatment, including the control, cells were treated with 1 mM IBMX 30 min prior to extraction and cAMP determination. A 15-fold increase of cAMP level was observed within the first hour of forskolin treatment (Figure 2). The increment level started to decrease gradually with time and reached 6-fold at the end of the study (48 h). In contrast, laminin-pretreated cells that were treated with DMSO did not show any detectable change in the cAMP level during a period of 48 h (Figure 3). These results suggest that laminin-induced cell differentiation may not be related to changes in the cellular cAMP level. On the other hand, the forskolin-induced cAMP increase may be important for the formation of long ramifying neurite-like processes.

Effect of forskolin and laminin on IRBP gene expression in WERI-Rb1 cells

To determine the effect of differentiating- and cAMP-inducing agents on the IRBP gene expression in WERI-Rb1 cells, laminin-pretreated cells were used for Northern blot analysis at different time intervals during forskolin treatment. As shown in Figure 4, there was a rapid and time-dependent increase in IRBP mRNA that reached a maximum of 3-fold at 6-8 h and returned to the basal levels after 24 h. In contrast, b-actin mRNA remained unchanged throughout the entire studying period. Therefore, the b-actin gene was used throughout the study as a reference for selective induction and for normalization of the IRBP mRNA levels.

To confirm the trend demonstrated by Northern analysis, the above study was repeated with RNase protection analysis with limited time course (0-24 h). IBMX was added 30 min prior to forskolin. As shown in Figure 5, IRBP mRNA increased rapidly to reach a maximum level of 2.7-fold at 2-4 h of treatment. Results showed that addition of phosphodiesterase inhibitor, IBMX, 30 min earlier than forskolin resulted in an earlier onset of IRBP gene induction but did not affect the magnitude of the induction (Data not shown)

To determine whether the modulation of the IRBP gene in differentiated WERI-Rb1 cells is cAMP-dependent, IRBP and b-actin mRNAs were determined with Northern blot analysis in the 3-day laminin-pretreated cells treated with DMSO for different periods of time. The result indicated that there was no significant increase in IRBP mRNA at any time (Figure 6). To demonstrate that the cAMP-dependent modulation of the IRBP gene requires laminin pretreatment, IRBP and b-actin mRNAs were determined with RNase protection analysis in cells pretreated with laminin for 1, 2, or 3 days and treated with forskolin/IBMX for 8 h. As shown in Figure 7, both IRBP and b-actin mRNA remained unchanged in controls and cells pretreated with laminin for 1 or 2 days. Only cells that were pretreated with laminin for 3 days showed a specific and significant increase in the IRBP mRNA. Taken together, these results suggest that 3 day laminin pretreatment and cAMP (induced by forskolin and stabilized by IBMX) are both required for the upregulation of the IRBP gene in WERI-Rb1 cells.

Effect of actinomycin D and cycloheximide on forskolin-induced IRBP gene expression in WERI-Rb1 cells

To probe into the mechanism of the cAMP-dependent IRBP mRNA induction, the half-life of the IRBP mRNA was examined by inhibiting mRNA transcription with actinomycin D in laminin-pretreated WERI-Rb1 cells. Cells were either treated with forskolin/IBMX or the solvent DMSO. Actinomycin D was added at a final concentration of 5 mg/ml at the beginning of the time course study. The disappearance of IRBP mRNA with time was measured by RNase protection assay. The results are shown in Figure 8. In the presence of actinomycin D, there was a significant reduction in IRBP mRNA in control cells and in cells treated with forskolin/IBMX. In contrast, there was no significant change in IRBP mRNA of the control cells in the absence of actinomycin D. These results suggest that the increased IRBP gene expression by forskolin in the laminin-pretreated cells was not due to the increased stability of the half-life of IRBP mRNA but was mainly due to the synthesis of IRBP mRNA.

To determine whether the above observed IRBP gene induction requires de novo protein synthesis, the above time course study was performed in the presence or absence of 10 mg/ml cycloheximide added at the beginning of the time course study. Within the time intervals from 0 to 8 h, the previously observed induction of IRBP mRNA was observed in the presence of cycloheximide (Figure 9). In the control cells, the expression of IRBP or actin gene was not affected by cycloheximide. This result suggests that the induction of the IRBP gene does not require de novo protein synthesis.

Discussion

The major finding of the current study is that the IRBP gene in differentiated WERI-Rb1 cells is selectively induced by cAMP. It is important to stress that this inductive effect of cAMP is observed only in laminin-induced, morphologically differentiated WERI-Rb1 cells. In morphological studies, we showed that the effect of cAMP was blocked by protein kinase A inhibitor. This suggests that the induction of the IRBP gene requires both laminin- and cAMP-mediated signal transduction pathway(s).

Laminin and its family members have been known to induce the differentiation of a number of cells [21]. The stimulatory effects of laminin have been attributed to activation of specific cell-surface laminin receptors. A 100 KDa cell surface laminin-binding protein has been partially characterized in the Y-79 retinoblastoma cells and thought to be a differentiating laminin-binding protein whereby laminin influences gene expression and development [22]. It is particularly significant that a relatively new homologue of laminin, S-laminin, a potent promoter of neurite outgrowth, has been found in the interphotoreceptor matrix in high concentration [23,24]. The temporal and spatial expression patterns of S-laminin during retina development suggest a role for this molecule in directing rod differentiation [25]. It is possible that laminin or S-laminin isoforms or combinations during early development are major determinants in photoreceptor maturation. Because the biochemical machinery required for expression of many photoreceptor genes is intrinsic to retinoblastoma cells [6,7], these cells may have direct relevance to early developing retina, and may provided a valuable model for the study of photoreceptor differentiation and development in vitro [21].

IRBP expression begins during the early stages of photoreceptor differentiation. To study photoreceptor differentiation, it is important to understand how the IRBP gene is activated during the early developmental stages. A previous study has demonstrated that the IRBP gene in the laminin-pretreated retinoblastoma cells is upregulated by cAMP after dbcAMP treatment for 5 days [9]. Given that controversy in the onset of IRBP gene activation and the fact that the butyrate can induce retinoblastoma cells [15], a time course study was required to assess the onset of IRBP gene activation. Forskolin, a direct adenylate cyclase activator, was used to eliminate the effect of butyrate itself. The phosphodiesterase inhibitor (IBMX) was used to protect the cAMP in the cells. We first determined the effect of forskolin on the levels of IRBP together with a reference mRNA during a time course of treatment. The results revealed that forskolin, a protein kinase A stimulating agent [26], increased the IRBP mRNA level in a time-dependent manner reaching a maximum of ~3-fold after 4-8 h and gradually decreased to basal level after 24 h (Figure 4 and Figure 5). It was noticed that the peak of IRBP mRNA level was reached at 2-6 h after the peak of cAMP level in retinoblastoma cells (Figure 2). Our data show that the enhancing effect of forskolin on cell differentiation was abolished by a protein kinase A inhibitor. The results also showed that the effect of forskolin on IRBP mRNA level can be detected only in cells treated with laminin for 3 days (Figure 7). Taken together, these results suggest that the molecular mechanism for the observed cAMP upregulation may be mediated by the protein kinase A-signaling pathway and laminin pathways.

In the previous reports that studied the modulatory effect of cAMP on the IRBP gene, it was not clear whether the IRBP gene is transcriptionally induced or the stability of its mRNA is increased by cAMP. It has been reported that genes that are rapidly upregulated by cAMP are transcriptionally induced [13]. On the other hand, genes that are slowly responsive to cAMP or insensitive to actinomycin D are likely mediated through a change in mRNA stability [14]. To clarify this point, we have repeated the time course study in the presence or absence of actinomycin D. The results demonstrate that the IRBP gene is selectively induced by cAMP and that the half-life of IRBP mRNA is not affected by cAMP-inducing treatments. Furthermore, the cycloheximide-insensitive induction of the IRBP gene by forskolin suggests the possibility of activation of latent transcription factors without de novo protein synthesis.

Genes that are transcriptionally induced by cAMP usually are regulated via a CRE-CREB and AP2 site-AP2 [13], or a CRS-Sp1 mechanism [27]. Based on sequence analysis and evidence that the IRBP gene is upregulated by cAMP, it was suggested that a region centered around -116 may contain a degenerate overlapping AP1/ATF-like element [28]. This region contains a CpG (Hpa II) site that undergoes hypomethylation during IRBP gene activation at the time of photoreceptor cell birth [20]. The IRBP gene also contains highly conserved G-rich motifs that are functionally active [29]. Additional work is required to determine the effects of laminin and cAMP on the IRBP promoter in order to define the mechanism of transcriptional regulation.

The importance of IRBP in the maintenance of the structural integrity of the photoreceptors has been demonstrated in IRBP knockout mice [4]. The cAMP-dependent induction of the IRBP gene is significant for the study of IRBP's function in photoreceptor development. As a fatty acid- and retinoid-binding protein in the interphotoreceptor matrix between the retinal pigment epithelium and photoreceptor cells [30,31], IRBP has been thought to play an important role in the extracellular trafficking of retinoids and fatty acids between these two types of cells. Although recent studies show that IRBP may not be involved in the visual cycle as a retinoid transporter [32,33], additional studies are required to determine the exact function of IRBP. The study of molecular mechanisms for the regulation of the IRBP gene should generate information advancing our knowledge on the molecular mechanisms underlying the processes of early photoreceptor development.

Acknowledgements

The authors thank Drs. B. Wiggert and G. Kutty for helpful comments on the manuscript, and Ms. Suraporn Matragoon for technical assistance. This work was supported by grant EY03829 from the National Institute of Health, Bethesda, MD, an Egyptian government scholarship, and an unrestricted Departmental award from Research to Prevent Blindness, Inc. to Medical College of Georgia.

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