a decrease in the polysomal population. - Proceedings of the National ...

A STABILIZATION OF RNA TEMPLA TES IN LENS CELL DIFFERENTIA TION* BY JAMES A. STEWARTt AND JOHN PAPACONSTANTINOU DEPARTMENT OF ZOOLOGY, UNIVERSITY OF CONNECTICUT, STORRS, AND BIOLOGY DIVISION, OAK RIDGE NATIONAL LABORATORY, OAK RIDGE, TENNESSEE

Communicated by Alexander Hollaender, May 2, 1967

There occurs in the vertebrate lens a specific stage of cellular differentiation during which the epithelial cell is transformed morphologically into an elongated fiber cell.' This process of cellular differentiation has been studied extensively by immunochemical,2 cytochemical,3-5 electron-microscopic,6' 7 and biochemical8' 9 techniques. These studies have shown that just prior to and during lens fiber cell formation, characteristic changes occur in the nucleus, nucleoli, and ribosomes.6' 7, 10, 11 Also, these studies indicate that significant changes in protein and nucleic acid synthesis occur during fiber cell formation. That these changes occur is further indicated by the observation that y-crystallin synthesis is initiated during fiber cell formation.2' 8 The differentiation of the lens epithelial cell to a fiber cell is an example of terminal cellular differentiation; this type of differentiation can also be seen in the maturation of the reticulocyte. A comparison of the two types of differentiating cells shows marked similarities: Maturation of the reticulocyte and lens fibrogenesis are accompanied by (a) loss of nuclear activity;3 (b) stabilization of mRNA;4' 9' 12 and (c) a decrease in the polysomal population.'3 It is the purpose of this paper to report on the stabilization of mRNA in the lens fiber cell and the effect of actinomycin D on protein synthesis in lens epithelial cells and fiber cells. Materials and Methods.-Calf eyes were obtained from the local abattoir and were placed on ice while being transported to the laboratory. The lenses were removed with their capsules intact and were placed in cold Hanks' salts. Lenses were preincubated in Hanks' salts for 10 min at 370C prior to the addition of amino acids from a C'4-labeled algal protein hydrolysate (0.5 Mc/ml) or uridine-2-C'4 (1.0 Ac/ml). Concentrations of actinomycin D were either 10 or 30 jug/ml. At the end of each experiment the lenses were washed with cold Hanks' salts. The epithelial cells were removed by removing the lens capsule. The remaining part of the lens (fiber cells) and the epithelial cells were immediately frozen (- 20'C) until needed. Preparation of lens protein homogenates: Homogenization was carried out in 0.005 M sodium phosphate buffer, pH 7. After centrifugation for 30 min at 12,000 g, the supernatant fraction was dialyzed for 16-24 hr with 1 volume change against 1 liter of 0.005 M sodium phosphate buffer, pH 7.0. DEAE-cellulose fractionation: The procedures for the fractionation of a-, As-, and y-crystallins on DEAE-cellulose columns and for identification of the eluted proteins have been described.8 14 DEAE-cellulose, type 20, was obtained from the Carl Schleicher and Schuell Co., Keene, N. H. All fractionations were carried out in a 50C cold room at flow rates induced by gravity. A NaH2 P04-Na2HPO4-NaCl buffer system ranging from pH 7-5.7 was used to elute the lens proteins from the DEAE-columns. Preparation of RNA by phenol extraction: Lenses were thawed and homogenized in 0.01 M Tris-HCl, pH 7.4, containing 0.01 M KCl, 0.0015 M MgCl2, polyvinyl sulfate (5 jg/ml) (PVS), and 1% sodium dodecyl sulfate (SDS). After homogenization, an equal volume of cold, watersaturated phenol (90% w/v) was added, and the preparation was shaken at 50C for 1 hr. The water-phenol emulsion was separated by centrifugation at 12,000 g for 30 min. The aqueous phase was removed and kept cold until the interphase material was extracted.

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Extraction of the interphase material was carried out by the method of McCarthy and Hoyer.'5 The interphase material was suspended in an equal volume of 0.02 M potassium acetate buffer, pH 5.0, containing 0.003 M MgCl9, 0.28 M LiCl, and 1% SDS. Two volumes of water-saturated phenol (90% w/v) were added, and the mixture was shaken at 60'C for 5 min. The hot phenol mixture was cooled to 4VC, and the emulsion was separated by centrifugation at 12,000 g for 30 min. The aqueous phases of the hot and cold phenol extractions were combined. The RNA was precipitated by the following procedure: The pH was adjusted to 5.2 with 0.2 N HCl. Potassium acetate was added to obtain a 2% solution, and then 2.5 volumes of cold 95% ethanol were added. The RNA was permitted to precipitate overnight at 0C. The precipitate was collected by centrifugation and dissolved in a small volume (1 to 5 ml) of 0.01 M Tris-HCl buffer, pH 7.4, 0.01 M KCl, 0.0015 M MgCl2, and 5 jsg/ml PVS. This solution was brought up to 250C and treated with DNase (5 JAg/mi) for 5 min. The DNase treatment was followed by another phenol extraction, and precipitated by the procedure described above. The precipitate was dissolved in Tris-HCl buffer, pH 7.4, 0.01 M KCl, 0.0015 M MgCl2, 5 pg/ml PVS, and dialyzed. This material was then used for sucrose density gradient analyses. Sucrose density gradient analyses: Resolution of the RNA was accomplished by using 26 ml of a linear, 5 to 20% sucrose (w/v) gradient layered over a 2-ml cushion of 60% sucrose (w/v). The gradients were made up in 0.01 M Tris-HCl, pH 7.4, containing KCl, MgCl2, and PVS in concentrations described above. The RNA sample was layered over the sucrose and centrifuged at 24,000 rpm for 16 hr at SoC in the Spinco model L2 preparative ultracentrifuge with the SW 25.1 head. After centrifugation, 1-ml fractions were collected from the bottom of the tube. The fractions were read for absorbancy at 260 mA. Analysis of radioactive fractions: Protein and RNA fractions were analyzed for radioactivity in a Nuclear Chicago Mark II liquid scintillation counter. In each case 0.5 ml of the fraction was dissolved in a hyamine-toluene-fluors mixture composed of 2 ml of a 1.5 M solution of Hyamine-10X (diisobutyl-cresoxy-ethoxy-dimethyl-benzyl-ammonium chloride monohydrate) in methanol plus 13 ml of a toluene solution of PPO (2,5-diphenyl-oxazole), 4 gm/liter and POPOP [p-bis-(5-phenyloxazolyl-2)-benzene], 50 mg/liter. All radioactivity is reported as disintegrations per minute.

Results.-The effect of actinomycin D on protein synthesis in the calf lens epithelial cells: Experiments were carried out to determine whether the synthesis of a-, l3-, and y-crystallins in the epithelial cells and fiber cells of the calf lens is inhibited by actinomycin D. Intact calf lenses were incubated at 37°C for 2 hr in the presence of C'4-algal protein hydrolysate with and without actinomycin D (10 ,ug/ml). Epithelial cells and cortex fiber cells were separated, and the a-, (3-, and 'y-crystallins from each cell type were fractionated on DEAE cellulose columns. An elution diagram showing the separation of a-, (3-, and y-crystallins extracted from control epithelial cells is shown in Figure 1. It can be seen here that there is a substantial degree of incorporation of amino acids into these proteins. By treatment of a group of lenses with actinomycin D, it was found that the incorporation of radioactive amino acids into epithelial cell proteins could be inhibited extensively. The elution diagram in Figure 2 shows a protein pattern similar to that in Figure 1. However, the specific activity (dpm/mg protein) was found to be significantly lower in the actinomycin-treated cells (Table 1). In an earlier report8 it was shown that lens epithelial cells do not contain -y-crystallins and that the synthesis of this group of proteins is specifically associated with fiber cell differentiation. However, the DEAE elution diagrams in Figures 1 and 2 do show significant quantities of y-crystallins in these cells. The presence of small amounts of y-crystallins in these epithelial cell homogenates is actually due to contamination by some elongating cells where the -y-crystallins first appear. From the data shown in Table 1 and Figures 1 and 2, it can be concluded that the synthesis

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FIG. 1.-Fractionation of calf lens epithelial cell crystallins. Regions of elution of the a-, 6B-, and -y-crystallins are designated by arrows. The solid line designates total protein (mg) per fraction; the broken line designates counts per minute per fraction. The proteins were eluted by a stepwise addition of the following buffers: (I) 50 ml of 0.005 M Na-phosphate, pH 7.0, (II) 50 ml 0.0075 M Na-phosphate, pH 6.5; (III) 50 ml 0.01 M Na-phosphate, pH 6.0; (IV) 75 ml 0.02 M Na-phosphate, pH 5.7; (V) 50 ml 0.02 M Naposphate, pH 5.7, +0.1 M NaCl; (VI) 50 ml 0.1 M Na-phosphate, pH 5.7, +0.1 M NaCl; (VII) 50 ml 0.1 M Na-phosphate, pH 5.7, +0.3 M NaCl. The fractions were collected in 3-ml aliquots; 78 rng protein were placed on the column, and 95% of the protein was recovered.

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FIG. 2.-Fractionation of calf lens epithelial cell crystallins from actinomycin D (10 ,sg/ml) treated lenses. Experimental conditions were the same as those described in Fig. 1; 78 mg protein were placed on the column, and 87% of the protein was recovered.

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of a-, (-, and y-crystallins in the cuboidal and elongating lens epithelial cells is potently inhibited by actinomycin D. The effect of actonomycin D on protein synthesis in lens fiber cells: Elution patterns showing the fractionation of a-, (-, and y-crystallins of lens cortex fiber cells incubated in the absence and in the presence of actinomycin D are seen in Figures 3 and 4, respectively. Both patterns are essentially identical with respect to the distribution of a-, (-, and y-crystallins. The degree of incorporation of amino acids into these lens proteins, however, is often observed to be significantly greater in the actinomycin-treated cells. A comparison of the specific activity of the a-, (3-, and -y-crystallins from control and actinomycin-treated lenses shows that there is a stimulation of protein synthesis (Table 1). These observations further indicate TABLE 1 OF ACTINOMYCIN D ON LENS ,Epithelial Cells (dpm/mg protein)+ Actinomycin D Inhib. Contro (10 Lg/ml) (%)

EFFECT

-y-Crystallins ct-Crystallins

fl-Crystallins

1590 963 1980

314 166 572

80 83 71

PROTEIN SYNTHESIS --Fiber Cells (dpm/mg protein)+ Actinomycin D Stimulation Control (%) (10 pg/ml)

235 99 340

396 165 690

68 66 103


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FIG. 3.-Fractionation of calf lens fiber cell crystallins. Experimental co were the same as those deanditions scribed in Fig. 1; 100 mg protein were placed on the column, and 87% of the pt rotein was recovered.

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that the protein synthesizing templates for y-crystallins (as well as for a- and crystallins)I in the fiber cell are insensitive to levels of actinomycin D which will produce an 80 per cent inhibition of the synthesis of this same group of proteins in the elongating epithelial cell. To determine whether the epithelial cells are required for a stimulatory effect, these cells were removed prior to incubation of the fiber cells with the C'4-amino acids. Under these conditions it was found that there was neither an inhibitory nor stimulatory effect on protein synthesis in the actinomycin-treated fiber cells. These data (Table 2) indicate that the localized stimulation of protein synthesis in the fiber cells may be dependent upon the presence of the epithelial cells. TABLE 2 EFFECT OF ACTINOMYCIN D ON LENS PROTEIN SYNTHESIS IN CALF CORTEX FIBER CELLS AFTER THE REMOVAL OF THE EPITHELIAL CELLS Expt. no.

(dpm/mg protein)

Control

Actinomycin D (dpm/mg protein)

1 2

29 50

34 37

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The stimulation of protein synthesis in calf fiber cells was found to vary greatly over a large number of experiments. Since the age of the calves from which lenses were obtained varied significantly, similar experiments were carried out with embryonic and adult lenses to determine whether the age of the lens is a factor in this stimulation. Through these studies it was found that actinomycin exerts a strong inhibition on crystallin synthesis in embryonic epithelial cells and that this inhibitory effect decreases as the age of the lens increases (Table 3). The data also show that in embryos and calves protein synthesis in the fiber cells is less susceptible to inhibition by actinomycin and that in the adult fiber cells protein synthesis is stimulated by the antibiotic. Thus, the variation of the effect of actinomycin on protein synthesis seen only in calf fiber cells (compare data in Tables 1 and 3) appears to be due to the age of these calves. The point that we wish to stress in this communication is that the differentiation of the lens epithelial cells to lens fiber cells at all ages involves a transition toward the stabilization of mRNA. TABLE 3 EFFECT OF ACTINOMYCIN D ON CRYSTALLIN SYNTHESIS IN EMBRYONIC, CALF, AND ADULT LENS CELLS Per Cent Inhibition (-) or Stimulation (+) in Fiber Cell

a

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In these experiments lenses were incubated in 30 pg/ml actinomycin D at 371C for 4 hr. were incubated in 10 pg/ml actinomycin D at 371C for 2 hr.

t Lenses

The effect of actinomycin D on RNA synthesis in calf lens epithelial cells and fiber cells: Since actinomycin D can inhibit the synthesis of lens crystallins in the epithelial cells and stimulate the synthesis of the same proteins in the more highly differentiated fiber cells experiments were carried out to determine whether RNA synthesis in these cells is affected by the antibiotic. Intact lenses were incubated at 370C for 2 hr in the presence of C14-uridine, with and without actinomycin D (10 pg/ml). RNA from epithelial cells and fiber cells was extracted by hot and cold phenol extractions. Sucrose density gradient analyses showed that there is a significant degree of incorporation of C14-uridine into the RNA (Fig. 5A) and that actinomycin D inhibits this incorporation in all fractions (Fig. 5B). This observation explains the potent inhibitory effect of actinomycin D on the synthesis of crystallins in the epithelial cells. Similar profiles for RNA from control and actinomycin-treated fiber cells are shown in Figures 6A and B. These data show that there is a sharp decline in RNA synthesis (compare Fig. 5A to Fig. 6A), and in the presence of actinomycin D the small amount of RNA synthesis observed in the control fiber cells is inhibited by more than 90 per cent. Thus, these studies indicate that (a) in the control fiber cells, when RNA synthesis is practically completely stopped, these cells maintain a significant rate of protein synthesis, and (b) under conditions in which RNA synthesis is potently inhibited in the fiber cells, protein synthesis in these cells may remain relatively unaffected or stimulated.


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FIG. 5.-(A) Sucrose density gradient profile of phenol-extracted RNA from calf lens epithelial cells. The lenses were incubated in the presence of uridine-2-C'4 for 2 hr as described in Materials and Methods. A260/ml 0 0 0; dpm/ml, 000; 15.5 OD,60 units were placed on the gradient. (B) Sucrose density gradient profile of phenol-extracted RNA from actinomycin-treated calf lens epithelial cells. Experimental conditions are the same as those described in (A); 15.0 OD260 units were placed on the gradient.

Discussion.-From the data presented, there is an indication that the RNA templates which regulate the synthesis of lens proteins are relatively more stable or long-lived in the highly differentiated fiber cell than in the epithelial cell. The existence of long-lived RNA templates has been detected in embryonic chick's and calf 16 lenses. In these experiments, however, the epithelial and fiber cells were not separated, and the degree of protein and nucleic acid turnover due to epithelial cells versus fiber cells could not be determined. From the studies presented here, it can be seen that since the epithelial cells differentiate to fiber cells, the initiation of stabilization of mRNA must occur at some time during fiber cell differentiation. A comparison of the specific activity of actinomycin-treated cells shows an inhibition of y-crystallin synthesis in the elongating epithelial cells and a stimulation of this same group of proteins in the fiber cells. Thus, at the time of appearance of y-crystallin (i.e., during the early stages of fibrogenesis)8 the synthesis of this protein, as well as of the a- and j3-crystallins, is still sensitive to actinomycin D, whereas in the completed fiber cell the synthesis of these same proteins is insensitive to the antibiotic and thus takes place on stable templates. It appears, therefore, that the stage of differentiation at which mRNA is stabilized can be localized at the latter stages of fiber cell formation or in the completed fiber cell. Fibrogenesis in the lens represents the final stage of cellular differentiation and results in the formation of a cell which has lost its ability to replicate. In this respect, the differentiation of lens cells is comparable to the maturation of the reticulocyte or to the maturation of skin cells. All of these cells have in common

VOL. 58, 1967


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