δ-Crystallin gene expression in embryonic chick lens epithelia cultured in the presence of insulin

Printed in Swden Copynyhf 0 1977 by Academic Press. Inc. in any form reserved A// rights of reproduction ISSN 00344827

Experimental

&CRYSTALLIN EPITHELIA

Cell Research 105 (1977) 9-14

GENE EXPRESSION IN EMBRYONIC CHICK LENS CULTURED IN THE PRESENCE OF INSULIN L. M. MILSTONE’

Laboratory

and J. PIATIGORSKY

of Molecular Genetics, National Institute of Child Health and Human Development, Bethesda, MD 20014, USA

SUMMARY Previous investigations have shown that the cells of the 6day-old embryonic chick lens epithelium elongate in defined medium supplemented with fetal calf serum or porcine insulin. In the presence of f&l calf serum, this cell elongation is associated with a differential increase in G-&ystallin synthesis which can be accounted for by accumulation of Gcrystallin messenger RNA (mRNA) during the 5th and 24th hours of culture.-We have examined whether S-crystallin gene expression is also regulated in the cultured lens epithelium when fetal calf serum is replaced by insulin. Embryonic lens epithelia cultured in insulin were labeled with [3Hfialine, and Gcrystallin was purified by high-resolution sodium dodecyl sulfate-gradient polyacrylamide gel electrophoresis; G-crystallin mRNA was quantitated by molecular hybridization with G-crystallin [%I]cDNA. The results showed that Gcrystallin synthesis increases and that Scrystallin mRNA accumulates in the lens epithelial cells between 5 and 24 h of culture in the presence of insulin. Stimulation of total protein synthesis, including &crystallin synthesis, occurred without an increase in G-crystallin mRNA content/cell during the first 5 h of culture with insulin, as was previously shown to occur during the first 5 h in vitro with fetal calf serum. Thus Gcrystallin synthesis is stimulated initially by an increase in translational efficiency and secondarily by an increase in the amount of G-crystallin mRNA when the embryonic lens epithelia are maintained in a chemically defined medium containing insulin.

Differentiation of chick lens epithelial cells into lens fiber cells can be studied in vitro when embryonic lens epithelia are cultured in medium containing fetal calf serum [ 1, 2, 31. Cell elongation and increased specialization for the synthesis of the lens-specific protein, b-crystallin, are the two most readily discernible features of this differentiation [4, 51. Previous work from this laboratory has shown that longitudinal alignment of microtubules correlates with cell elongation [6], and that accumulation of Scrystallin messenger RNA (mRNA) is associated with increased specialization for b-crystallin synthesis [7].

Our interest in the effect of insulin on cultured lens cells began when a report appeared showing that insulin caused microtubule assembly in cultured rat adipocytes [8]. Subsequent studies from our laboratory demonstrated that 1 pg/ml of insulin was as effective as fetal calf serum in promoting cell elongation [9] and microtubule alignment [lo] in the cultured embryonic chick lens epithelium. Those experiments also suggested that insulin was not as effective as fetal calf serum in stimulating protein synthesis in the cultured embryonic chick lens epithelium [lo]. We have subsequently reexamined this question in greater detail Exp

Cc/[

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utilizing improved culture and radioactive labeling conditions. We have employed a r3H]DNA complementary to &crystallin mRNA (b-crystallin cDNA) [II] to quantitate by molecular hybridization the effect of insulin on &crystallin mRNA production by the cultured lens epithelia. The results establish that insulin, like fetal calf serum, promotes a differential increase in the rate of 6-crystallin synthesis, which is associated with accumulation of 6-crystallin mRNA in the elongating cells of the cultured lens epithelia. MATERIALS

AND METHODS

Cell counts The number of cells in trypsindissociated thelia was counted as previously [5].

SDS-polyacrylamide

lens epi-

gel electrophoresis

Aliquots of the SDS homogenates representing 2-3 dissolved explants were heated to 100°C for 3 min in the oresence of 1% B-mercaptoethanol and electropho;esed for 18 h at’75 V and room temperature in discontinuous 10 cm slab gels of 7.5-30 % linear gradients of polyacrylamide containing 0.1% SDS, at pH 8.9; an approximate 1 cm stacking gel of 3% polyacrylamide containing 0.1% SDS, at pH 6.8, overlaid the separating gel and a Tris-glycine buffer at pH 8.9 was used in the reservoirs [ 151.After electrophoresis, the gels were stained with 0.2% Coomassie Brilliant Blue (Eastman Chemical Co.) in 9% acetic acid and 45 % methanol followed by electrophoretic destaining in 7 % acetic acid and 5 % methanol. G-Crystallin was readily identified in the gels as the protein band which had a molecular weight of 50 000 [ 161and which comigrated with purified 8-crystallin, electrophoresed in a oarahel slot of the gel slab. The Gcrystallin band was cut from the gel, di&olved in H,O,, and assayed for radioactivity in a Beckman LS 233 liquid scintillation spectrometer using a toluene-based scintillation fluid containing 8.7 % Bisolv BBS-3 (Beckman Instruments).

Tissue culture and [3H]valine labeling of proteins The central regions of lens epithelia from 6-day-old White Leghorn chick embryos (obtained from Truslow Farms, Inc., Chestertown, Md) were explanted into 30 mm tissue culture dishes (Falcon Plastics) and cul[3H]valine specific tured at 37°C in a humidified atmosphere of 5% CO%, Intracellular 95% air in 2 ml of Ham’s F-10 culture medium [12] activio determinations supplemented with 1 pg/ml of porcine insulin (Eli Lilly Co.) as described elsewhere [5]. The amount of CO2 The snecific activity of intracellular [3H]valine in the lens epithelia was determined by analysis of picomolar in the tissue culture incubator was carefully controlled amounts of [‘“C]dansylj3H]valine isolated by twobv analvsis with a Fvrite CO, indicator (Bacharach dimensional thin-layer chromatography, as described Ir&umknt Co.). The indicator revealed that our in detail by Milstone & Piatigorsky [5]. previous tests [IO] were actually conducted at approx. 2.5 % CO2 rather than 5 % CO,. The insulin concentration used is optimal for promoting lens cell elongation Synthesis and hybridization of in these explants [9]. After the indicated times in vitro, &crystallin [3H]cDNA 18-24 explants were placed into conical polyethylene tubes (Be1 Arts Plastics) in 0.25 ml of fresh medium &Crystallin [3H]~DNA was synthesized on a template supplemented with 1 pg/ml of insulin and 80 &i/ml of of purified d-crystallin mRNA with avian myelor3H]valine (SchwarzlMann, 16 Cilmmole) and mainblastosis virus reverse transcriptase and fractionated tained in the tissue culture incubator for 60 min. The by alkaline sucrose density-gradient centrifugation, as tissues were then washed once with Saline G [13], described in detail elsewhere [ 1I]. The [3H]cDNA used twice with acetone at -2O”C, and dissolved in 10 PI/ in these experiments was from a single preparation exalant of 0.01 M Tris, uH 7.5, containing 0.14 M consisting of molecules 900-I 100nucleotides in length NaCl and 1% sodium ‘dbdecyl sulfate (SDS); this with an auoroximate specific activity of 10’ cpm/pg preparation was called an SDS-homogenate. Aliquots RNA : [?I]cDNA hybridizations were performed in were taken for scintillation counting to determine in80 ~1. oil-sealed reaction mixtures containing 2000 corporation into total proteins, for RNA extraction, cpm of [3H]~DNA, total RNA from 8 tissues, 10 mM for DNA determination, and for SDS-polyacrylamide Tris-HCI (uH 7.5). 0.6 M NaCl, 0.2 mM NaEDTA and gel electrophoresis. 2 pg of single-stranded salmon sperm DNA (Sigma Chemical Co.). The lens cell RNA was extracted from the SDS-homogenates with phenol : chloroform : isoDNA determinations amyl alcohol as previously [7]. Twenty ~1 aliquots were removed after 0, I, 2 and 3 h incubation at 75°C. The DNA from aliauots of the SDS-homogenates representing an equivalent of l-2 dissolved explants was Hybrid formation was assayed by resistance to digestion by the S1 nuclease from Aspergiilus otyzae as nrecinitated overnight at -20°C with 75% ethanol. previously described [7]. The 0 time sample was the DNA was measured by the diamino benzoic acid fluobackground subtracted from the other samples, and rescence method of Kissane & Robbins [ 141in a Bowgenerally contained less than 3% nuclease resistant man Aminco spectrofluorometer using calf thymus radioactivity. DNA (Sigma Chemical Co.) as a reference. Exp Cell Res 105 (IY77)

Gene expression and insulin

11

cell returned to the original level by 24 h in lens explants cultured in the presence of vitro; by contrast, the rate of r3H]valine incorporation into b-crystallin/cell continued insulin” to increase and was 2.3 times higher after Cell no. X 10m3 Time 24 h of culture than at the time of explantaDNA in vitro (cells/ (ngkpithelium) epithelium) (hours) tion. The specific activity of intracellular r3H]23.7t0.5 88f 7 0 valine was determined in separate experi113f15 31.6kl.l 20.4kO.6 93_+ 7 2: ments [5]. Epithelia were preincubated for 0, 5 and 24 h and labeled for 60 min in mea Average of 4 expts +S.E.M. dium containing r3H]valine. The measured specific activity of intracellular [3H]valine was the same for each preincubation time RESULTS and averaged 1 100 Ci/mole. The possibility Cell number and DNA content in lens that the extracellular pool of [3H]valine was explants cultured in the presence of insulin utilized for protein synthesis instead of the In order to normalize the data on protein intracellular pool has been considered elsesynthesis and b-crystallin mRNA content in where [5]. In that case, too, the precursor the cultured lens epithelia, the number of pool would have the same specific activity cells and DNA content of the explants were for each set of epithelia. Thus, since the precursor specific activities were the same determined immediately after explantation, after 5 h in vitro and after 24 h in vitro (ta- and newly synthesized &crystallin is not ble 1). There was an approximate 1.25-fold degraded in cultured lens cells [lo, 5, 181, average increase in the cell number and comparisons of valine incorporation into 6DNA content of the epithelia during the crystallin are equivalent to comparisons of first 5 h of culture. By 24 h in vitro, these S-crystallin synthesis. values were approximately the same as the original values, probably reflecting a detachment of some cells from the lens epitheTable 2. Protein synthesis and &crystallin lium under these conditions. In view of this mRNA content in lens explants cultured in finding, DNA determinations were performed on every sample which was ex- the presence of insulin” r3H]Valine incorporation amined for protein synthesis and &crystalin 1 h lin mRNA content in order to accurately Total proteinb Wrystallin’ Krystallin Time normalize the results. Table 1, Cell number and DNA content of

Protein synthesis in lens explants cultured in the presence of insulin

Experiments with r3H]valine showed that there was a 1.6-fold stimulation of incorporation into total proteins and into Gcrystallin, per lens cell, after 5 h of culture in the presence of insulin (table 2). The rate of C3H]valine incorporation into total proteins/

in vitro (hours)

(cpdng DNA)

(cpmhg DNA)

mRNA” (pglng DNA)

0 5 24

150+ 16 236+20 173+ 15

2Ok3 31f3 45f2

2.6kO.2 2.8kO. 1 9.4kO.8

a Average of 4 experiments ?S.E.M. * Aliquots of the SDS-homogenate. ’ Krystallin purified by SDS-polyacrylamide gel electrophoresis. d RNA from SDS-homogenates hybridized to b-crystallin [3~]~~~~. E.vp Cdl

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and Piatigorsky

t

20t 10

t

oL--1

5

10

20

L

10-a

10-3

Fig. 1. Abscissa: (A) Number of epithelia times hours of hybridization; (B) RJ, which is defined as A,/ml divided by 2 times hours of hybridization; ordinates: % hybridization of the [3H]~DNA, which is defined as the fraction of the [3H]~DNA resistant to S, nuclease times 100. (A) These three curves from a single experiment represent the % of krystallin [3H]cDNA hybridized to RNA from lens epithelia previously cultured in insulin for 0, 0; n , 5 and A, 24 h. The points on each curve represent different lengths of time for the hybridization reaction. These results are not adjusted for differences in cell number; (B) this curve represents a standard hybridization reaction of purified krystahin mRNA with its [3~]~~~~. The molar ratio of mRNA to cDNA was 4.5. Calculations of the amount of Scrystallin mRNA in the tissue extracts (A) by reference to this standard curye showed that the molar ratios of mRNA to cDNA were approx. 2 for the 0 and 5 h extracts and 6 for the 24 h extract. Since in control experiments we have found, at most, very small effects on- hybridization by small changes in- the mRNA : cDNA ratios, as in the range used in these experiments, the product of log (mRNA in tissue extract) x time which gives 40% hybridization should equal the product of log (purified krystallin mRNA) x time which gives 40% hybridization. See text for further comment.

&Crystallin mRNA content in lens explants cultured in the presence of insulin

Previous investigations have established the purity and specificity of our Ccrystallin [3H]cDNA and have demonstrated its usefulness as a probe for quantitating b-crystallin mRNA by molecular hybridization [7, II]. The &crystallin [3H]~DNA utilized in the present experiments was 900 to 1 100 Exp

Cell

Res IO5 (1977)

nucleotides long, so that a minimum of 1526% of its nucleotide sequence must encode the translated region (1500 nucleotides) of 6-crystallin mRNA, which has a total length of 2 200 nucleotides [ 111. The amount of RNA per lens explant which hybridized to a constant amount of b-crystallin [3H]~DNA in a given time increased during the 24 h of culture of the epithelia in the presence of insulin (fig. 1A). The b-crystallin [3H]~DNA did not appreciably hybridize with RNA extracted from headless bodies of chick embryos of the same stage of development [7], demonstrating the specificity of the hybridization reaction. These hybridization data can be converted to the number of 6-crystallin mRNA molecules present in the lens explants by reference to a standard hybridization reaction performed with known amounts of purified 6-crystallin mRNA under the same conditions (fig. 1B). Since the hybridization kinetics of the experimental (fig. 1A) and the standard reaction (fig. 1B) were comparable, namely the slopes of the hybridization reactions were the same, we conclude that the different mRNA: cDNA ratios present in the different tests did not significantly affect the hybridization reaction rates, and that we were justified in using the standard curve for quantitating the 6-crystalline mRNA content of the lens explants (see caption to fig. 1 for further discussion of this point). After normalization for the differences in cell number in the explants, the data showed that there was actually the same number of Gcrystallin mRNA molecules per cell at the time of explantation and after 5 h in vitro, and that there was 3.6 times more &crystallin mRNA per cell in the explants after 24 h of culture in the presence of insulin (table 1). An additional experiment (data not shown) in which as-

Gene expression and insulin

says were performed every 5 h showed an approximately linear rate of accumulation of b-crystallin mRNA per cell between 5 and 25 h of culture.

13

while in the present tests the explants were cultured in an atmosphere containing 5 % CO,. Since the pH of the culture medium (Ham’s F-10) is determined by the pC0, of the atmosphere, the lower COZ concentration would result in a more alkaline meDISCUSSION dium, especially in the absence of serum The present experiments demonstrate an proteins which help to buffer the medium. increased rate of &crystallin synthesis and Small increases in alkalinity markedly dian accumulation of 6-crystallin mRNA in minish the adherence of epithelial cells to 6-day-old embryonic chick lens epithelial the lens capsule (unpublished observations) cells cultured for 24 h in defined medium and could well explain the loss of cells from supplemented with porcine insulin. Com- the explants cultured in Ham’s F-10 meparable increases take place in these cells dium supplemented with insulin. (2) In our cultured for 1 day in the presence of fetal previous experiments [lo], the lens explants were cultured in medium containing insulin calf serum [5, 71. Although the b-crystallin mRNA measurements in this study used but, in contrast to the present tests, were RNA extracts from whole cells, we be- radioactively labeled in medium lacking inlieve these are reasonable estimates of the sulin. The combination of a higher pH and amount of b-crystallin mRNA in the cyto- labeling without insulin possibly created less optimal culture and labeling conditions plasm, since a previous study of &crystallin mRNA in epithelia cultured in the presence than existed in the present set of experiof fetal calf serum demonstrated that the ments. nuclei contained only 1% of the total celSince the explanted embryonic lens epilular &crystallin mRNA [7]. In contrast to thelial cells elongate [9] and develop lonb-crystallin synthesis, the rate of total pro- gitudinally oriented cytoplasmic microtein synthesis in the lens explants is not tubules [lo] in medium supplemented with higher after 24 h of culture in the presence insulin, as they do in medium supplemented of insulin than at the time of explantation, with fetal calf serum [4], it is apparent that implying a differential increase in the rate of morphological and biochemical features &crystallin synthesis and a lowering in the characteristic of lens fiber cell differentiarate of synthesis of other cellular proteins. tion will occur in vitro in a chemically deSuch a non-coordinate control of protein fined medium containing insulin. It is unsynthesis also occurs in these lens epithelial likely that trace amounts of glucagon or cells during the first 24 h of culture in me- zinc contaminating the insulin preparation dium containing fetal calf serum [5, 181. are responsible for the increased rate of 6Several factors may be responsible for crystallin synthesis or 8-crystallin mRNA our earlier results suggesting that b-crystal- accumulation in the cultured explants, inaslin synthesis is not elevated in the lens ex- much as these do not stimulate lens cell plants cultured in medium supplemented elongation in the absence of insulin [9]; with insulin [lo]. (1) As noted under Mate- moreover, reduced and carboxymethylated rials and Methods, in our previous experi- A and B chains of insulin also fail to elicit ments the lens explants were cultured in an lens cell elongation in these explants [6]. atmosphere containing approx. 2.5 % COB, The idea that both insulin and serum pro-

14

Milstone and Piatigorsky

mote a pleiotypic response from cultured cells [19] is supported by our observations that the complex series of events associated with lens fiber cell differentiation occurs in embryonic lens epithelia cultured in the presence of insulin or fetal calf serum. It is interesting to note that, in contrast to the differentiative response of the cells of cultured 6-day-old embryonic chick lens epithelia subjected to serum or insulin immediately after explantation, serum or insulin starvation of the explants for several days cause the cells to divide rather than differentiate upon subsequent serum [20] or insulin (unpublished observations) addition. Furthermore, the epithelial cells of cultured 19-day-old embryonic chick lens epithelia [21] and of cultured adult mammalian lenses [22, 231divide rather than differentiate into lens fibers in response to serum or insulin. We find that the rates of &crystallin synthesis and total protein synthesis increase in the cultured lens epithelial cells during the first 5 h in vitro in medium containing either fetal calf serum [7] or, as the present investigation shows, insulin, without an increase in the amount of &crystallin mRNA/ cell. This finding is similar to results with cultured fibroblasts in which the stimulation of a general increase in protein synthesis by serum or hormones is regulated at the level of mRNA translation [see 241. It differs from the differential increase in &crystallin synthesis between the 5th and 24th h of culture which appears to be governed by the amount of 8-crystallin mRNA present in the cytoplasm. Further work is required to determine whether during the first 5 h of culture insulin or fetal calf serum causes each b-crystallin mRNA molecule to be

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translated more efficiently or increases the number of S-crystallin mRNAs being translated . We thank Dr Peggy Zelenka for the cDNA, Dr Philip Leder for the reverse transcriptase and S, nuclease, Eli Lily and Company for a gift of the insulin, and Mrs Cecilia Levi for expert preparation of the manuscript.

REFERENCES 1. Philpott, G W & Coulombre, A J, Exp cell res 38 (1%5) 635. 2. Piatigorsky, J, Rothschild, S S & Milstone, L M, Dev hiol 34 (1973) 334. 3. Piatigorsky,‘J, Dev biol(1974) f 21. 4. Piatigorsky, J, Webster, H deF & Craig, S P, Dev biol27 (1972) 176. 5. Milstone, L M & Piatigorsky, J, Dev biol43 (1975) 91. 6. Piatigorsky, J, Ann NY acad sci 253 (1975) 333. 7. Milstone, L M, Zelenka, P & Piatigorsky, J, Dev biol48 (1976) 197. 8. Soifer, D, Braun, T & Hechter, 0, Science 172 (1971) 269. 9. Piatigorsky, J, Dev biol30 (1973) 214. 10. Piatigorsky, J, Rothschild, S S & Wollberg, M, Proc natl acad sci US 70 (1973) 1195. 11. Zelenka, P & Piatigorsky, J, Exp eye res 22 (1976) 115. 12. Ham, R G, Exp cell res 29 (1%3) 515. 13. Puck. T T. Cieciura. S J & Robinson, A, J exp med iO8 (1958) 945. 14. Kissane, J M & Robins, E, J biol them 233 (1958) 184. IS. Maize], J V Jr, Methods in virology 5 (1971) 179. 16. Piatigorsky, J, Zelenka, P & Simpson, R T, Exp eye res 18 (1974) 435. 17. Zelenka, P & Piatigorsky, J, Proc natl acad sci US 71 (1974) 18%. 18. Beebe,DC &Piatigorsky, J, Expeyeres22(1976) 237. 19. Hershko, A, Mamont, P, Shields, R & Tomkins, GM, Nature new biol 232 (1971) 206. 20. Philpott, G W, Exp cell res 59 (1970) 57. 21. Piatieorskv. J & Rothschild, S S, Dev biol 28 (197T) 382: 22. Harding, C V, Reddan, J R, Unaker, N Y & Bagchi, M, Int rev cytol 31 (1971) 215. 23. Reddan, J R, Unakar, N J, Harding, C V, Bagchi, M & Saldana, G, Exp eye res 20 (1975) 45. 24. Shields, R, Nature 258 (1975) 194. Received June 30, 1976 Accepted October 20, 1976