Hormonal regulation of myosin heavy chain and alpha-actin gene expression in cultured fetal rat heart myocytes.

Thyroid hormone regulates the expression of ventricular myosin isoenzymes by causing an accumulation of alpha-myosin heavy chain (MHC) mRNA and inhibiting expression of beta-MHC mRNA. However, the mechanism of thyroid hormone action has been difficult to examine in vivo because of its diverse actions. Accordingly, hormonal control of expression of six MHC isoform mRNAs and cardiac and skeletal alpha-actin mRNAs was studied in primary cultures of fetal rat heart myocytes grown in defined medium. The results indicate that in the absence of thyroid hormone, cultured heart cells express predominantly beta-MHC and cardiac alpha-actin mRNAs. Addition of 3,5,3'-triiodo-L-thyronine (T3) caused a rapid induction of alpha-MHC mRNA and decreased beta-MHC mRNA levels without affecting the skeletal muscle MHC mRNAs. There was an almost parallel change in the myosin isoenzymes. Cardiac alpha-actin mRNA levels were transiently increased by T3 treatment, but skeletal alpha-actin was unaffected. Elimination of insulin and epithelial growth factor from the medium did not alter the effects of T3 on cardiac MHC mRNA expression. Addition of various adrenergic agents to the medium had no appreciable effect on cardiac MHC mRNA expression despite the presence of functionally coupled alpha- and beta-adrenergic receptors. Addition of steroid hormones, muscarinic agents, and glucagon to the medium also had no effect. Thus, under defined conditions, T3 is able to regulate MHC gene expression at a pretranslational level without the need for other exogenous factors.


Hormonal Regulation of Myosin Heavy Chain and a-Actin Gene Expression in Cultured Fetal Rat Heart Myocytes*
(Received for publication, February 2, 1987) Thomas A. GustafsonSQlI Thyroid hormone regulates the expression of ventricular myosin isoenzymes by causing an accumulation of a-myosin heavy chain (MHC) mRNA and inhibiting expression of j3-MHC mRNA. However, the mechanism of thyroid hormone action has been difficult to examine in vivo because of its diverse actions. Accordingly, hormonal control of expression of six MHC isoform mRNAs and cardiac and skeletal a-actin mRNAs was studied in primary cultures of fetal rat heart myocytes grown in defined medium. The results indicate that in the absence of thyroid hormone, cultured heart cells express predominantly &MHC and cardiac a-actin mRNAs. Addition of 3,5,3'-triiodo-~-thyronine (T3) caused a rapid induction of a-MHC mRNA and decreased &MHC mRNA levels without affecting the skeletal muscle MHC mRNAs. There was an almost parallel change in the myosin isoenzymes. Cardiac aactin mRNA levels were transiently increased by T3 treatment, but skeletal cr-actin was unaffected. Elimination of insulin and epithelial growth factor from the medium did not alter the effects of T3 on cardiac MHC mRNA expression. Addition of various adrenergic agents to the medium had no appreciable effect on cardiac MHC mRNA expression despite the presence of functionally coupled a-and &adrenergic receptors. Addition of steroid hormones, muscarinic agents, and glucagon to the medium also had no effect. Thus, under defined conditions, T3 is able to regulate MHC gene expression at a pretranslational level without the need for other exogenous factors.
Myosin, a major component of the contractile apparatus in striated muscles, is a hexamer consisting of two heavy chains (molecular mass -200 kDa) and two pairs of light chains (molecular masses -16 and 20 kDa). Myosin heavy chain (MHC)' isoforms are known to be encoded by a highly con-served multigene family (1,2). Seven MHCs are known to be expressed in striated muscles of the rat, including embryonic and perinatal skeletal muscle isoforms, two mature fast skeletal muscle forms, one form expressed only in extraocular muscles, and two cardiac isoforms, a and P (3). Expression of these genes has been shown to be subject to developmental (3,4) and innervational regulation (5, 6). In addition, all of the MHC genes expressed in rat striated muscles are regulated by thyroid hormone in a highly complex manner (7)(8)(9).
In ventricular muscle, the a-and P-MHC isoforms can combine to form three isoenzymes termed V1-V3, in order of decreasing electrophoretic mobility in nondenaturing gels. The V1 and V3 forms are comprised of two a-MHCs and two P-MHCs, respectively, whereas the Vp form is made up of one a-MHC and one P-MHC each (10). These myosin forms have the same light chain composition, but differ in enzymatic activity. The Vl form has the highest ATPase activity, the V3 form the lowest, and the Vz form an intermediate level of activity (11). Because myosin ATPase activity is closely related to the speed of contraction in heart and skeletal muscles (12)(13)(14), the effects of thyroid hormone on myosin isoenzyme composition may explain, in part, the ability of thyroid hormone to stimulate cardiac performance (15).
Thyroid hormone regulates the expression of ventricular myosin isoenzymes by stimulating synthesis of a-MHC mRNA and inhibiting expression of (3-MHC mRNA (8,16,17). Similar alterations in MHC isoenzyme expression by thyroid hormone have also been observed in cultured rat heart cells (18). The a-and P-MHC genes are linked in tandem 4 kilobase pairs apart in a 5' to 3' orientation that corresponds to the order of their developmental expression in ventricular myocardium (19). The inverse manner in which these genetically linked genes are regulated by thyroid hormone provides a particularly interesting system for study of hormonal regulation.
In addition to changes during development and in response to alterations in thyroid status, a number of other neurohumoral factors have been implicated in control of cardiac MHC expression, including sex steroids (20), glucocorticoids (21), and catecholamines (22). Because of the many interactions of thyroid hormone with other endocrine systems (23,24), cardiac cell culture systems have been sought that would permit the study of MHC regulation under more defined conditions. Suitable permanent cell lines derived from heart muscle are unavailable, but some success has been reported using primary heart cell cultures. Nag and Cheng (18) demonstrated that thyroxine causes increases in the Vl myosin form and decreases in the V3 form in primary fetal heart cell cultures grown in serum-free medium. In the present study, we have examined the effects of thyroid hormone and a number of drugs and neurohumoral substances on MHC and a-actin mRNA expression in primary cultures of fetal rat heart cells grown in defined medium. To assess more directly the effects of thyroid hormone on MHC and a-actin gene expression, we have measured mRNA levels by use of a sensitive dot-blot assay based upon synthetic oligonucleotide probes designed to be complementary to the unique 3'-untranslated regions of the MHC and a-actin mRNAs.

EXPERIMENTAL PROCEDURES
Materials-Earle's salt solution and pancreatin were purchased from GIBCO. Ham's F-12K culture medium was obtained from KC Biologicals, Lenex, KS. A premixed solution of insulin, transferrin, and selenium (ITS) was purchased from Collaborative Research, Inc. Synthetic oligonucleotide probes were prepared in an Applied Biosystems DNA synthesizer (Model 380A). 32P-Labeled r-ATP was purchased from New England Nuclear; my0-[2-~H]inositol (15.0 Ci/ mmol) was purchased from Amersham Corp. Culture plates were obtained from Falcon, Oxnard, CA. All other drugs and chemicals used in these experiments were purchased from Sigma.
Cell Culture-Primary cultures of cardiomyocytes were prepared essentially as described by Nag and Cheng (18) with minor modifications. Hearts from 18-day fetal rats were minced and digested in Earle's salt solution, without Ca2+ and M$+, containing 0.125% pancreatin. After discarding the initial two 15-min digests, cells were collected and resuspended in serum-containing medium (25). After complete digestion, the cells were resuspended in Ham's F-12K medium containing 250 pg/ml fetuin, 10 mg/ml bovine serum albumin, 20 pg/ml ascorbic acid, 100 units/ml penicillin, and 100 pg/ml streptomycin (pH 7.4). The cells were plated for 3 h to allow fibroblast attachment. Unattached cells were pelleted and resuspended in the culture medium to which was added 20 ng/ml epithelial growth factor, 5 pg/ml insulin, 5 pg/ml transferrin, and 5 ng/ml selenium. The final yield of cells was 1.5-2 X lo6 cells/fetal heart. The cells were plated at a density of 2.5-3.0 X lo6 cells/plate onto 100-mm Petri dishes that had been coated with a solution containing 0.1 mg of rat tail collagen and 80 pg of fibronectin in 5 ml of Earle's salt solution. Stock solutions of T3 were prepared in 0.1 N NaOH and diluted in culture medium before use. Steroids were dissolved in 50% methanol, and all other drugs were dissolved directly in culture medium. Control plates treated with diluent gave results that were not distinguishable from untreated controls. Cultures generally consisted of greater than 90% myocytes as measured by periodic acid-Schiff staining for glycogen (26). Over 90% of the cells began to contract spontaneously within 24 h after plating.
RNA Preparation and Dot-Blot Assays-After removal of the medium, the cells were scraped directly into 1 ml of guanidinium isothiocyanate solution using a rubber policeman, and total cellular RNA was prepared using the guanidinium isothiocyanate/hot phenol procedure (27). All plastic and glassware were pretreated with 0.1% diethylpyrocarbonate at 37 "C for 30 min and autoclaved. The final RNA pellet was resuspended in distilled HzO, quantitated by its absorbance at 260 nm, and either spotted immediately or stored at -70 "C. Levels of MHC and a-actin mRNAs were measured by use of a dot-blot assay as described earlier (7,8). In brief, synthetic oligonucleotide probes 20 bases in length were purified by electrophoresis and labeled at the 5' end using T4 polynucleotide kinase and [r-?P]ATP. An additional oligonucleotide probe was synthesized whlch hybridizes to a region of common sequence in the coding region of the a-and P-MHC mRNA immediately upstream from the termination codon (5'-CGCCAATGTCACGGCTCTTG-3') (28). Percent changes in myosin mRNAs were calculated as follows: ((cpm of drug treated platescpm of untreated control plates)/cpm of untreated control plates) X 100.
Analysis of Myosin Isoenzymes-Myosin was prepared from cultured myocytes using methods similar to those described earlier (18,29). Myocyte cultures were washed with an ice-cold solution containing 40 mM NaCl and 3 mM Na2HP04 (pH 7.0) and scraped directly into 0.5 ml of extraction buffer containing 10 mM Na4P20, (pH 8.8), 5 mM EGTA, 15 mM 0-mercaptoethanol, 1 mM phenylmethanesulfonyl fluoride, 2 pg/ml leupeptin, and 2 pg/ml pepstatin. The solution was homogenized in a prechilled Dounce homogenizer and kept on ice for 1 h with occasional mixing. The samples then were centrifuged at 100,000 X g for 1 h. The supernatant was collected and analyzed for protein using bovine serum albumin as a standard (30). An equal volume of cold glycerol was added to the supernatant, which was used for electrophoresis immediately or stored at -70 "C. No differences were observed in the electrophoretic pattern between fresh and stored myosin samples.
Myosin isoforms were separated on polyacrylamide gels using the methods and apparatus described earlier (10). Myosin samples (5-10 pg) were loaded directly onto gels that had been prerun for 30 min at 90 V. Electrophoresis was carried out at 90 V for 20-24 h at 2-4 "C. The gels were stained in a solution containing 0.04% Coomassie Brilliant Blue R and 3.5% perchloric acid and destained in a mixture of 7.5% acetic acid and 5% methanol. The gels were analyzed by densitometry using a microprocessor-based spectrophotometer (Response, Gilford Instrument Co., Oberlin, OH).
Phosphatidylinositol Turnover and CAMP Assays-For [,H]inositol-labeling experiments, cells were cultured for 1 day in Ham's F-12K medium at a density of about 5 x lo5 cells/well in 24-well plates.
At the end of this time, cells were labeled for 21 h with my0- [2-~H] inositol at a specific activity of 2 x lo-* pCi/pmol and a final concentration of 100 p~ (31). Cells then were rinsed and incubated in fresh medium for 5 h. Lithium (10 mM LiCl) was added to the medium 5 min before the addition of selected drugs to prevent recycling of phosphorylated inositol derivatives to inositol. All media used for treated cells contained 10 nM Tg, and control cells were grown in media containing diluent throughout the experiment. The incubation was terminated after 60 min by rinsing the cells three times with 0.5 ml of ice-cold buffer containing 137 mM NaC1,4.7 mM KCl, 1 mM MgSO, 1.3 mM CaCl,, 1.5 mM M&&, 3.24 mM NazHPO,, 0.76 mM KHZPO4, 12.5 mM Hepes, and 5 mM glucose (pH 7.4). After aspiration of the wash buffer, 0.5 ml of methanol was added to each well, and the contents were transferred to a chloroform-resistant tube containing 1 ml of chloroform and 0.5 ml of HZO. A second aliquot of 0.5 ml of methanol was added to each well and combined with the original extract. After vortexing, the tubes were centrifuged for 15 min at 2700 rpm, and the aqueous phase was assayed for [3H]inositol phosphate by the method of Berridge et al. (31) as modified (32).
Cyclic AMP formation experiments were carried out with cells grown for 2 days in the presence or absence of 10 nM T,. After 1 min preincubation with 0.625 mM 3-isobutyl-l-methylxanthine, the cells were incubated for 3 min with selected drugs and placed on ice. The media then were removed, 150 p1 of buffer containing 4 mM EDTA and 50 mM Tris-HC1 (pH 7.4) was added, and the wells were scraped. The contents of each well were transferred to microcentrifuge tubes, which were placed in boiling HzO for 5 min to denature proteins. Following centrifugation, the supernatants were analyzed for CAMP by the competitive binding assay (33) as modified (32). Statistical A~lyses-Intergroup comparisons of experimental data were made by the Student-Newman-Keuls procedure (34) after an analysis of variance had indicated significant differences. This procedure was implemented on a microcomputer using the statistical package SPSS/PC+ (SPSS).
Differences between experimental groups were accepted as statistically significant at the p < 0.05 level.

RESULTS
Expression of MHC and a-Actin Isoforms in Cultured Myocytes-Dot-blot analysis of MHC and a-actin oligonucleotide probes with RNA from cultured myocytes is shown in Fig. 1.
In the absence of T3, significant hybridization was observed only with probes for P-MHC and cardiac a-actin. Addition of T3 to the media for 48 h caused a marked induction of a -MHC mRNA levels and a simultaneous decrease in the level of P-MHC mRNA. A slight increase in the expression of cardiac a-actin mRNA was also observed. Probes for fast IIa, fast IIb, embryonic, and perinatal MHC mRNAs did not hybridize with RNA from cultured cells either before or after T3 treatment. Thus, cultures of 18-day fetal myocytes retain their ability to phenotypically express the cardiac-specific MHC and a-actin isoforms, and upon T, administration they are able to switch from a predominance of P-MHC mRNA to the a-MHC form.
Kinetics of Thyroid Hormone Effects-The kinetics of changes in the levels of cardiac MHC and a-actin mRNAs after addition of 10 nM T3 are shown in Fig. 2. Hybridization of RNA prepared from cultures after  h of treatment with the a-MHC probe revealed a rapid increase in a-MHC to levels 10-15 times greater than that found in untreated  MHC mRNA is consistent with an early increase in a-MHC mRNA levels before /3-MHC mRNA begins to decline and suggests that cardiac myocytes maintain tight control over total MHC mRNA levels. Interestingly, cardiac a-actin mRNA levels also increased after Ts addition by about 200% (Fig. 2) but then rapidly returned to control levels.
The effects of Ts on cardiac myosin isoenzymes are shown in Fig. 3. The isoenzyme pattern observed after an initial overnight attachment period in the absence of T3 was found to be approximately 75% V3 and 25% VI. After 4 days in culture without T3 treatment, the cells almost exclusively expressed the V3 form. By contrast, addition of thyroid hormone to the medium caused a rapid change in the myosin isoforms from a predominance of the V3 form to the VI form. The increase in VI was apparent after only 24 h of treatment. By the third day of T3 treatment, the isoenzyme pattern was nearly the inverse of that found in untreated plates with approximately 8 0 % VI and 20% V3. The close correspondence between changes in cardiac myosin isoenzymes and MHC mRNAs suggests that regulation of the MHC genes occurs a t a pretranslational level.
Dose-Response Relationship of Thyroid Hormone Concentration to MHC Switching-The dose-response relationships for the effect of TS treatment on cardiac MHC mRNA levels is shown in Fig. 4 Analysis of Effects of Media Components on MHC SMitching-Requirements for control of cardiac MHC expression were analyzed further by selective omission of the growth factors which were routinely added to the media, that is epithelial growth factor and a solution of insulin, transferrin, and selenium. As shown in Fig. 5, omission of either one of these components or both had no effect on induction of a-MHC mRNA or repression of 8-MHC mRNA by T3. These results clearly demonstrate that thyroid hormone acts directly on myocytes to mediate changes in cardiac MHC mRNAs. Although the addition of these growth factors was not necessary for MHC switching, when they were omitted from the media, RNA yields dropped by about 30%. Consequently, they were included in all subsequent experiments.
Effects of Cardwactiue Drugs and Neurohumoral Factors on MHC Expression-Percent changes from untreated controls after treatment with T3, various drugs, and the combination of T3 plus drug are shown in Table I. Statistical analysis of the data used to calculate these changes showed that all T3treated cultures were significantly different (p < 0.01) from untreated and drug-treated controls. No significant interactions were observed between T3 and drug treatments.
Thus, addition of norepinephrine, isoproterenol, or phenylephrine at a concentration of 1 p~ or 1 mM dibutryl CAMP to T3-treated myocytes had no significant effects on the degree of a-MHC mRNA induction or 8-MHC mRNA repression compared with T3 treatment alone. Similarly, when these agents were administered to cells in the absence of T3, levels of MHC mRNA were similar to those observed in cells cultured in the absence of all drugs. To determine whether there were any additive effects between thyroid hormone and these  A number of other agents were examined in a similar manner, including carbamylcholine chloride (carbachol), corticosterone, testosterone, estradiol, and glucagon at a concentration of 1 PM. None of these compounds were found to significantly affect a-or P-MHC expression in the absence or presence of T3.
Because it has been postulated that contraction of the myocyte may be involved in regulation of myosin expression (35), the effects of T3 in the presence of K' (50 mM KCl) were examined. As shown in Table   I, the patterns of thyroid hormone regulation by T3 in arrested cells were similar to those observed in beating cells except that P-MHC mRNA counts/min were less than the untreated control plates in the absence of T3. Nevertheless, the patterns of a-MHC induction and p-MHC repression remained intact, suggesting that contraction is not an essential requirement for MHC switching.
Assays for Neurohumoral Receptors-In view of the apparent lack of effect on MHC gene expression observed upon addition of adrenergic agents to the medium, assays were performed to determine whether the fetal myocytes contained functionally coupled &adrenergic and muscarinic receptors.
The effect of isoproterenol on the stimulation of cAMP formation is shown in Fig. 6. Formation of cAMP was assayed after addition of graded concentrations of isoproterenol in cells grown for 48 h in the absence or presence of 10 nM T,. Although the EC6, in the presence of T3 (23 nM) was slightly lower than the value obtained in its absence (34 nM), after subtraction of the basal activities, the two dose-response curves were quite similar. As shown in Table 11, maximal cAMP stimulation could be almost completely blocked with propranolol. These results indicate that functional P-adrenergic receptors were present in these cells.
To determine whether functional al-adrenergic receptors were present in the cells, norepinephrine-induced PI turnover was analyzed by the formation of my~-[~H]inositol l-phosphate in the presence of 10 mM LiCl (32). As shown in Fig. 7, the concentration of norepinephrine which stimulated PI turnover to 50% of the maximal stimulation above baseline (ED,,) was 1.3 PM in the absence of T3 and 0.76 PM in the presence of T,. Furthermore, PI turnover induced by either norepinephrine (1 FM) or phenylephrine (1 PM) could be inhibited by 1 PM prazosin (Table 111). These results suggest  that the cultured myocytes contained functional a,-adrenergic receptors.
To test for the presence of functional muscarinic receptors, the effect of M carbamylcholine on isoproterenol-induced cAMP production was examined. Inverse coupling to @-adrenergic receptors was demonstrated by a 65% reversal of isoproterenol stimulation of cAMP production in the absence of T, and a 56% reversal in the presence of T3 (data not shown).

DISCUSSION
In these experiments, regulation of cardiac MHC and aactin expression has been studied in primary cultures of fetal rat heart myocytes under defined conditions. Patterns of MHC and a-actin expression in response to thyroid hormone seemed to be similar to those found in the intact hypothyroid ventricle (8). In the absence of TS, P-MHC mRNA was the predominant isoform expressed, whereas the presence of T3, rapid induction of a-MHC mRNA expression occurred with repression of P-MHC mRNA. The demonstration of an EC50 of 1-2 nM T3 for a-MHC induction and p-MHC repression, which is similar to the reported plasma free-T3 level in the euthyroid rat (1.5 nM) (36), strongly suggests that the hormonal regulatory mechanisms observed in culture are physiologically relevant. Furthermore, the effects of thyroid hormone did not require the presence of neurohumoral substances or addition of other exogenous factors. Contrary to recent data suggesting that glucocorticoids (21), sex steroids (20), or al-adrenergic receptors may control P-MHC gene expression (37), T3 was the only substance tested that affected expression of either the a-or P-MHC genes, suggesting that it may be the primary physiological regulator of these genes. Alternatively, it is possible that the regulatory mechanisms through which agents other than T3 act in vivo are not present at this early stage of cardiac development or are lost upon culturing. Although the existence of functional a-and padrenergic receptors is evident in these cells, assays were not available for the functional analysis of the other agents.
Earlier studies of myosin isoform expression in cultured cells have been limited to quantitation of the protein isoforms. The availability of a specific dot-blot assay for each MHC mRNA permitted the determination of whether embryonic, neonatal, or fast skeletal isoforms are expressed in these cells. Interestingly, none of the other MHC mRNAs were expressed either in the absence or presence of T3. These results indicate that the MHC gene family is subject to at least two types of regulation. One type involves the stable repression of MHC genes within a particular muscle type in a manner that makes them unresponsive to alterations in thyroid status. The second mechanism involves active regulation of the MHC gene dependent upon thyroid status. Both of these mechanisms seem to be retained in the cultured heart cells.
Although skeletal a-actin has been shown to be expressed in neonatal rat ventricle (38), in the current study, 18-19-day fetal cardiomyocytes did not express significant levels of this mRNA after several days in culture. Because the medium used was serum-free, it is possible that continued expression of the skeletal isoform in culture may require the presence of additional factors (39). Cardiac a-actin mRNA was constitutively expressed in the absence of T3, but could be transiently stimulated during the first 2 days of treatment with T3. A similar phenomenon has been observed in the hearts of hypothyroid rats treated with thyroid hormone (8). Under the culture conditions used in the present experiments, thyroid hormone treatment did not cause induction of the skeletal aactin mRNA in cultured myocytes, even though skeletal aactin mRNA apparently can be induced in rat heart under conditions of pressure overload (40). The close correlation between the changes in cardiac MHC mRNA and their respective protein isoenzymes suggests that the mechanism of thyroid hormone action on the MHC genes is a t a pretranslational level. The fact that a-MHC mRNA is induced from negligible basal levels also is consistent with a transcriptional control mechanism. Furthermore, pretranslational control is suggested by the recent demonstration that thyroid hormone can induce expression of an a-MHC/chloramphenicol acetyltransferase fusion gene containing only the 5' region of the a-MHC gene, after transfection into primary myocytes (41). However, the experimental results cited do not entirely rule out concomitant alterations in mRNA processing and stability.
It is interesting to note that the time course of induction of the a-MHC mRNA occurs somewhat in advance of P-MHC repression (Fig. 3). Although this may indicate some differences in the control of these two genetically linked genes, it also may simply reflect the time necessary for p-MHC mRNA breakdown to occur after mRNA production has slowed. The almost identical dose-response relationships observed with a-MHC induction and fl-MHC repression would seem to favor similar control mechanisms.
The analysis of the effects of media components on MHC mRNA switching showed that thyroid hormone is sufficient to mediate all aspects of cardiac MHC gene regulation in primary fetal myocyte cultures. Furthermore, addition of adrenergic and muscarinic agents caused no appreciable effect on MHC gene expression, even though functionally coupled adrenergic and muscarinic receptors were demonstrated. These results suggest that thyroid hormone probably plays a dominant role in the regulation of MHC expression in vitro.
Despite this finding, there is evidence to suggest that a number of other factors participate in the regulation of MHC expression in uiuo independent of thyroid status. For example, myocardial hypertrophy induced by pressure or volume overload is associated with rapid increases in &MHC mRNA and decreases in a-MHC mRNA in the rat ventricle which cannot be explained by alterations in thyroxine levels (42). Interestingly, these changes could be reversed by high-dose thyroid hormone administration but not by physiological replacement doses.
Most of the biological effects of thyroid hormone are believed to be mediated through a nuclear receptor-T3 complex (43). Recently, two groups of investigators have presented data to suggest that the c-erb-A gene, the cellular counterpart of the viral oncogene v-erb-A, may be a nuclear receptor for thyroid hormone (44,45). Homologies to the steroid hormone receptors also were found, particularly in the putative DNA binding domain. Other recent evidence suggests that the nuclear T3 receptors may bind directly to specific regions of certain genes under thyroid hormone control, such as those encoding growth hormone and placental lactogen (46). These lines of evidence suggest that the mechanism of thyroid hormone action may resemble that proposed for the steroid hormones, a process involving binding to its specific nuclear receptor, followed by an interaction with DNA sequences in the 5'-flanking sequences of regulated genes which alters their transcription. The cultured cell system described in this study would seem to be quite suitable for further dissection of these regulatory processes.