Androgen regulates the level and subcellular distribution of the AU-rich ribonucleic acid-binding protein HuR both in vitro and in vivo.

HuR, a member of the ELAV family of AU-rich RNA-binding proteins, is present in a variety of tissues and is directly involved in stabilizing labile AU-rich messenger RNAS: We have found that treating the human HepG2 cell line with 10 nM dihydrotestosterone (DHT) for 48 h decreases the total level of HuR by 75%. DHT decreases both cytosolic and nuclear HuR levels in HepG2 cells, but increases HuR levels in polyribosomes by 325%. In BALB/c mice, HuR levels in the submaxillary salivary gland (SMG) and the kidney display a dramatic sexual dimorphism, but those in the spleen and thyroid do not. DHT (200 microg) causes total HuR levels in female SMG and kidney to fall progressively, whereas, conversely, orchiectomy of males causes HuR levels to rise in these two tissues by 800% and 200%, respectively. As an internal control we probed the same blots for AUF1, a destabilizing AU-binding protein, and confirmed our previous findings showing that the cytosolic p37 isoform of AUF1 shows the opposite responses of cytosolic HuR in the SMG, and that the level of AUF1 in the kidney does not respond to DHT. In polyribosomes from female mouse SMG, HuR levels doubled after 6 h of DHT, but decreased by 80% after 24- and 48-h DHT treatment. Thus, the total level of HuR is regulated in two different androgen-responsive systems, as is the shuttling of HuR between different subcellular compartments. As AUF1 is responsive to androgen in the mouse SMG, but not in the kidney, tissue-specific posttranscriptional regulation of AU-rich messenger RNA metabolism could be mediated in part by differential androgen-dependent regulation of HuR and AUF1.

Inhibiting proteasomes in human HepG2 and LNCaP cells increases endogenous androgen receptor levels.

Treating HepG2 cells with MG132 for 4 h to inhibit proteasomal activity increased androgen receptor immunoreactivity in two major bands with molecular weights of 102 and 110 kDa by 77% each (P < 0. 05). MG132 treatment also increased the overall level of polyubiquitinated proteins between 66 and 220 kDa by 140% (P < 0.05). Antiubiquitin immunoreactivity comigrating with the androgen receptor bands was also increased by MG132 treatment. Two other proteasome inhibitors, lactacystin and epoxomycin, caused similar increases in the androgen receptor in HepG2 cells. Proteosome-inhibition studies conducted in LNCaP cells also showed that the two major androgen receptor bands with molecular weights of 102 and 110 kDa were increased by 85 and 115%, respectively (P < 0. 05 for both) by MG132 treatment. Overall levels of polyubiquitinated proteins with molecular weights between 66 and 220 kDa increased 365%. Ubiquitin immunoreactivity comigrating with the androgen receptor bands was also significantly increased. Thus inhibiting proteasomes in two human androgen-responsive cell lines increases endogenous androgen receptor levels as well as androgen receptor-associated ubiquitin-modified immunoreactivity. The regulation of steady-state levels of endogenous androgen receptor by proteasomal degradation could be involved in its rapid turnover in the absence of ligand and would provide a mechanism for limiting androgen responses. A PEST sequence similar to one in the vitamin D receptor is present in the hinge region of all known mammalian androgen receptors, suggesting that it may function in proteasome-mediated androgen receptor turnover.

The internalization and endosomal trafficking of the EGF receptor in response to EGF is delayed in the waved-2 mouse liver.

The EGF receptor in waved-2 mice contains a point mutation that renders it kinase-deficient. We investigated how the waved-2 mutation affects the internalization and endosomal trafficking of the receptor in vivo in response to EGF. When the waved-2 mice were injected with EGF, there was approximately 50% less tyrosine phosphorylation detected in whole-liver homogenate compared to wild-type background mice. Although EGF increased the EGF receptor levels in the early and late liver endosomal fractions of waved-2 mice, its trafficking was delayed compared to wild-type mice. Ubiquitination of the EGF receptor may affect its endosomal sorting. We found that a similar amount of EGF receptor was immunoprecipitated from the endosomal fractions of EGF-treated waved-2 and wild-type with anti-ubiquitin antibody. These results demonstrate that the waved-2 EGF receptor can become ubiquitinated and can be trafficked to the late endosomes, although it appears that its kinase deficiency delays this process.

Testosterone and dihydrotestosterone regulate AUF1 isoforms in a tissue-specific fashion in the mouse.

The sex difference in the metabolism of certain mRNAs in the murine submaxillary gland (SMG) prompted us to determine whether androgens regulate the expression of any of the four isoforms of AUF1, proteins that bind differentially to AU-rich RNA. We found that cytosol from female SMGs contains two major isoforms (p45 and p40), whereas cytosol from male SMGs contains a prominent p37 and a weaker p42. Injecting female mice with testosterone decreases p45 levels by 81% after 7 days (P < 0.05, n = 4), whereas p42 and p37 increase 74 and 449% at 7 days (P < 0.05, n = 4, for both). Orchiectomy, conversely, decreases p37 levels in the male SMG by 91% (P < 0.006) while increasing p45 5-fold and p40 2.5-fold (P < 0.05, n = 5 for both). Both male and female kidney cytosol contains a prominent p37 and a faint band of approximately 42 kDa, but neither shows a significant change when circulating androgen levels are altered. Dihydrotestosterone (DHT) changes the pattern of AUF1 isoforms in female SMG cytosol more rapidly than does testosterone. Nuclear extracts from female SMG contain predominantly p45, and DHT decreases its level slightly (35%, P < 0.05 at 24 h). Polysomal extracts from female SMG contain p45 and p42, and DHT increases p45 levels 58% (P < 0.02, n = 6) at 24 h. In certain nonreproductive tissues, androgens may differentially regulate AUF1 isoform levels to modulate the metabolism of AU-rich mRNAs posttranscriptionally.

Thyrotropin regulates the levels of free ubiquitin and ubiquitin-protein conjugates in the male mouse thyroid.

The conjugation of ubiquitin to proteins can be a signal for their degradation, but can also be involved in regulatory processes not directly involved in protein degradation. Because thyrotropin (TSH) is the major physiological regulator of the thyroid, we have investigated whether changes in the circulating level of TSH influence the level of immunoreactive ubiquitin in the thyroid, as assessed by dot-blot assays. Putting male Balb/c mice on a low iodine diet with methimazole (MMI) in their drinking water for 14 days raised the level of ubiquitin by 425 % (p < 0.001) per microgram nonthyroglobulin protein (the mean thyroglobulin level dropped by 35% (p < 0.05)). Western blots similarly indicated that immunoreactivity migrating in the region of monoubiquitin, ubiquitin oligomers, and ubiquitinated thyroid proteins increased on the low iodine/MMI diet. Injecting 1 microg triiodothyronine (T3) 6 hours prior to sacrifice appeared to reduce the ubiquitin levels by 31% when compared with mice only on the low iodine/MMI (p < 0.07), but injection of T3 had no effect on ubiquitin levels in mice on the control diet. Injecting male ICR mice with a large dose of TSH (200 mU) increased immunoreactive ubiquitin levels by 50% (p < 0.05) 2 hours later. After a second dose of TSH was injected 12 hours later, the level of immunoreactive thyroglobulin fell by 17% (p < 0.05). With further 12 hourly injections, thyroglobulin levels then began to reaccumulate, and they had returned to the level of saline-injected controls after 50 hours (five TSH injections), while ubiquitin levels fell, but remained significantly elevated above the saline-injected controls (36%, p < 0.01). When ICR mice were given perchlorate in their drinking water to block the iodide pump, thus preserving thyroidal responsiveness to repeated TSH injections, the responses to the initial two TSH injections were similar to mice who received ordinary tap water. However, with further TSH injections, the reaccumulation of thyroglobulin did not occur: at 50 hours, thyroglobulin levels remained suppressed by 28% (p < 0.05), and ubiquitin levels actually rose slightly, and were significantly higher (57%, p < 0.01) than the saline-injected controls. These in vivo responses of free ubiquitin, oligoubiquitin, and ubiquitinated protein to changes in the level of circulating TSH suggest that ubiquitin-mediated mechanisms are involved in some of the thyroid's metabolic responses.

The cytosolic 47-kilodalton protein that binds to the 3' untranslated region of epidermal growth factor transcripts responds to orchiectomy in a tissue-specific fashion.

The submaxillary gland (SMG) of the male mouse is known to contain more epidermal growth factor (EGF) messenger RNA (mRNA) than that of the female. In contrast, the SMG of female mice contains more of a cytosolic 47-kDa RNA binding protein that binds to a unique 23-b sequence at the end of the 3' untranslated region (3' UTR) of EGF, which includes the canonical polyadenylation signal in an AU-rich region containing a potential mRNA destabilizing sequence. Testosterone treatment can change the activity of this 47-kDa protein within 1 day, which precedes the change produced in the level and in the polyadenylation of EGF mRNA in female mice. In the kidney, however, neither the 47-kDa protein nor the polyadenylation pattern changed after testosterone. We now report that 2 weeks following orchiectomy of Balb/c mice, mature EGF peptide immunoreactivity levels fall by 96% (P < 0.0002), and mRNA levels fall by 76% (P < 0.0001). Orchiectomy also enhances the RNA binding activities of the 47-kDa protein in SMG cytosol but not in the kidney: UV cross-linking to 3' UTR RNA is increased by 67% (P < 0.01) and shifting of the gel mobility of the RNA is increased by 47% (P < 0.05). The polyadenylation pattern of EGF transcripts also changes in SMG but not in kidney cytosol. After orchiectomy, the population of EGF transcripts in the SMG with short, heterogenous poly-A tails (<50 A's) decreases by 31% (P < 0.03), whereas transcripts with long poly-A tails of approximately 50, 70, 100, and 200 A's all increase and become more distinct, resembling the pattern found in the normal female. Thus, 2 weeks after orchiectomy, the responses in EGF expression, EGF mRNA polyadenylation, and the activities of the 47-kDa 3' UTR binding protein are the reciprocal of the responses produced by injecting female mice with testosterone for 5 days, providing further evidence that circulating androgens regulate EGF expression posttranscriptionally in a sexually dimorphic, tissue-specific fashion.

Testosterone regulates tissue-specific changes in the binding of a 47-kilodalton protein to a highly conserved sequence in the 3' untranslated region of epidermal growth factor messenger ribonucleic acid.

Epidermal growth factor (EGF) transcripts that use the terminal polyadenylation signal display a dramatic sex difference in the pattern of polyadenylation in the murine submaxillary gland (SMG), whereas those in the kidney do not. It takes 3 days before testosterone treatment begins to change the polyadenylation pattern in female SMG to resemble the male pattern, a finding that supports previous suggestions that posttranscriptional mechanisms are involved in regulating EGF expression. The conservation of a unique 23-b sequence centered on the terminal polyadenylation signal in all published mammalian EGF sequences suggested that trans-acting factors involved in EGF messenger RNA (mRNA) metabolism might bind to this sequence. To investigate this, we prepared 32P-RNA containing the 3' terminal EGF 23-b sequence plus a short poly-A tail, and incubated it with SMG cytosol. Cytosol retarded the electrophoretic mobility of this RNA as a single prominent band on 8% PAGE, and by UV-cross-linking, a single prominent 47-kDa protein was detected on 10% SDS-PAGE. Trypsin abolished both the gel-retarding and cross-linking activities. Cytosol from female SMGs contained approximately 8 times more of both the RNA binding activities than male cytosol. Injecting testosterone (200 microg QOD) into female mice altered both the RNA binding activities in a biphasic fashion, initially increasing them by about 40% at 2 days, then decreasing them by about 65% > or = 5 days, reaching male levels. Kidney cytosol contained both RNA binding activities but displayed neither sexual dimorphism nor testosterone-responsiveness. The tissue-specific testosterone-dependent changes observed in the 47-kDa protein occur before the increase in EGF mRNA levels and before the change in EGF mRNA polyad-enylation, so this cytosolic protein could be a trans-acting factor involved in EGF polyadenylation.

Increased epidermal growth factor expression produced by testosterone in the submaxillary gland of female mice is accompanied by changes in poly-A tail length and periodicity.

The level of mature EGF messenger RNA (mRNA) in the female submaxillary salivary gland (SMG) begins to rise after 3 days' treatment with testosterone (200 micrograms sc qod), and by 5 days it reaches a plateau of approximately 5 times baseline. Testosterone can increase the transcription of other genes in the SMG rapidly, so the lag in the EGF is not due to a slow androgen receptor response, and EGF mRNA can respond rapidly to other mediators, so the lag is not an innate characteristic of EGF transcription. Immunoreactive EGF levels reach a steady-state several times greater than the plateau reached by EGF mRNA, suggesting that testosterone also enhances the efficiency of EGF mRNA translation. Because testosterone has been reported to alter poly-A polymerase activities and because the translation and stability of some mRNAs is affected by changes in their polyadenylation, we used 3' rapid amplification of complementary DNA ends (3' RACE) to determine whether testosterone affected this aspect of EGF RNA metabolism. We found that EGF transcripts in untreated female SMG occur indistinct size classes, with poly-A tails of approximately 20, 50, 70, 100, and 200 A's attached after the terminal polyadenylation signal. In contrast, EGF transcripts in male SMG have poly-A tails of less clearly defined lengths, being more heterogeneous, ranging from approximately 20-100 A's. Treating female mice with testosterone causes the poly-A pattern in the SMG to change to a more heterogeneous population ranging from approximately 20-100 A's, similar to the male pattern. This change in EGF transcript polyadenylation occurs concurrently with the changes observed in the levels of EGF mRNA. EGF transcripts from male or female kidney contain distinct poly-A tails of approximately 20, 50, 70, 100, and 200 A's: neither EGF mRNA levels nor polyadenylation was altered by testosterone. The tissue-specific increase in EGF mRNA levels and in translational efficiency produced by testosterone in the female mouse SMG could involve this posttranscriptional alteration in transcript polyadenylation.

Assessment of transcript polyadenylation by 3' RACE: the response of epidermal growth factor messenger ribonucleic acid to thyroid hormone in the thyroid and submaxillary glands.

The complementary DNA (cDNA) sequence of epidermal growth factor (EGF) indicates that its 3' untranslated region (3' UTR) is 745 bases long, with polyadenylation occurring at residue 4749. However, when we used reverse transcriptase-polymerase chain reaction (RT-PCR) with an anchored 3' primer [3'rapid amplification of cDNA ends (RACE)] to amplify the 3' ends of cDNA, we actually detected two major products [800 and 600 base pairs (bp)] and a minor product (400 bp) in the thyroid or submaxillary glands (SMGs) of male mice. Analysis of genomic DNA with a battery of primer pairs gave only the predicted PCR products from the 3' UTR, demonstrating the lack of introns in this region of genomic DNA and eliminating alternate splicing as the explanation of the transcript diversity we detected. We confirmed that two potential polyadenylation sites proximal to residue 4749 are used in vivo by hybridizing the same 3' RACE products with probes specific for the 5' end of the 3' UTR, and also for poly-A tails. To assess the distribution of poly-A tail lengths on transcripts using the terminal polyadenylation site (4749), we used several different approaches to analyze 3' RACE products. Solution hybridization with 3' UTR probes revealed a striking difference between transcripts in SMG and thyroid: SMG contained two large 3' RACE populations (approximately 770 and 870 bp), whereas thyroid only contained one (approximately 770 bp). EGF transcript heterogeneity due to different poly-A tail lengths was confirmed using an upstream primer 400 bases closer to the 3' end of the 3' UTR, and TaqI digestion. Again we found two major populations in SMG (approximately 380 and 480 bp), but only one (380 bp) in thyroid, which upon TaqI digestion showed tissue-specific heterogeneity only in the 3' fragment. T4 treatment of male mice (0.25 microgram T4/gm ip) increased the intensity of both populations in SMG and the smaller population in thyroid within 24 h. However, after a week of T4 injections, only the intensity of the population with the longer poly-A tails in the SMG remained elevated, a finding consistent with tissue-specific enhanced stability of transcripts due to polyadenylation. Finally, to resolve poly-A tail lengths more precisely, we used an upstream primer that was specific for the 3' end of murine 3' UTR. This approach revealed that the thyroid contains three major populations of EGF transcripts, with poly-A tail lengths of approximately 20, 50, and 70 A's. After T3 treatment for 24 h, the intensity of transcripts containing 20 A's increased 52% (P < 0.02) and those with 50 A's increased 130% (P < 0.01), whereas there was no change in transcripts with tails > or = 70 A's. On the other hand, there were no distinct bands in SMG samples, but rather a heterogeneous distribution of poly-A tail lengths from approximately 20-120 A's that showed an overall increase of approximately 60% in response to T3.

Secondary structure in the 3' UTR of EGF and the choice of reverse transcriptases affect the detection of message diversity by RT-PCR.

The secondary structure in mRNA is essential for many processes, but it can present a technical problem in making full-length cDNA with reverse transcriptases. Furthermore, different reverse transcriptases have differing abilities to transcribe through regions with secondary structure, which can alter the products obtained by reverse-transcribing RNA and then PCR-amplifying the product (RT-PCR). We have been interested in studying the posttranscriptional regulation of epidermal growth factor by RT-PCR and have tested the ability of several reverse transcriptases to reverse transcribe the 3'-untranslated region (3'UTR), a region that contains substantial secondary structure. When low levels of either total RNA or poly(A)+ mRNA were used, we found avian myeloblastosis virus reverse transcriptase (AMV-RT) to be the most robust of all the enzymes tested. Furthermore, contrary to reports that AMV-RT is inhibited by tRNA--which should make it less effective than Moloney murine leukemia virus reverse transcriptase (MMLV-RT) at reverse-transcribing total RNA--adding tRNA to poly(A)+ RNA actually increased the amount of specific RT-PCR product obtained with AMV-RT while it decreased the amount of product and enhanced mispriming with MMLV-RT. We found that pre-incubation of the oligo(dT) primer with total RNA at elevated temperature prior to reverse transcription improved the efficiency of both native and modified MMLV-RTs. These findings support the concept that secondary structures in RNA differentially affect the abilities of different reverse transcriptases to detect transcript diversity and raise the possibility that such structures could affect quantitation using RT-PCR with internal mRNA standards.

The ways in which hormones change cyclic adenosine 3',5'-monophosphate-dependent protein kinase subunits, and how such changes affect cell behavior.

Hormones and cytokines regulate many cellular functions by activating the ubiquitous cAMP-dependent protein kinase (A kinase) system. Newly synthesized cAMP molecules bind to regulatory (R) subunits in A kinase holoenzymes, causing them to release their catalytic (C) subunits. These free C subunits then phosphorylate proteins until the cAMP level falls, whereupon the R subunits regain their affinity for free C subunits, and thus form inactive holoenzymes again. However if cAMP levels remain persistently elevated, many cells change their A kinase system. Some cells alter the rate of degradation of subunits, and some cells change the level or stability of the messages encoding subunits. Cellular behavior often changes if cAMP levels remain elevated: many cells differentiate, some cells proliferate, and some cells die, depending on the stage of the cell cycle. The two forms of A kinase holoenzyme (type I and type II) contain identical C subunits, but contain either an RI dimer or an RII dimer. In some tissues, type II holoenzyme is compartmentalized to subcellular organelles via specific anchoring proteins, whereas type I holoenzyme is generally cytosolic. Free RI subunits turn over more rapidly than free RII subunits in most cells, but all free subunits are degraded more rapidly than when they are associated together in holoenzymes. Free C subunits can phosphorylate a broad spectrum of proteins in both the cytoplasm and nucleus, depending on the type of cell, its state of differentiation, and the hormonal milieux. If free C subunit is microinjected into the cytoplasm of some intact cells, it migrates to the nucleus, whereas if free R subunit is microinjected, it remains in the cytoplasm. If both subunits are coinjected, R subunit blocks the nuclear migration of the C subunit. A major nuclear target for free C subunits is the CREB family of nuclear proteins, which bind to cAMP response elements (CREs) in the promoter regions of cAMP-responsive genes. Phosphorylation of CREB proteins alters their ability to form dimers and to interact with CREs. Many CREB proteins can be phosphorylated by other kinases as well, indicating this is one means by which cells coordinate cAMP- and non-cAMP-mediated gene responses. However, interactions between CREB and a number of other nuclear proteins with which they can dimerize, especially proteins whose levels are rapidly altered in response to hormones, provide an even higher degree of complexity of gene regulation than is possible from various kinases phosphorylating the different sites in CREB proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Thyroxine increases the levels of epidermal growth factor messenger ribonucleic acid (EGF mRNA) in the thyroid in vivo, as revealed by quantitative reverse transcription polymerase chain reaction with an internal control EGF mRNA.

Treating mice with T4 increases the level of immunoreactive epidermal growth factor (EGF) in the thyroid. In order to establish whether this response might reflect a T4-dependent increase in the levels of messenger RNA (mRNA) in the thyroid, we prepared an internal standard which permits us to quantitate EGF mRNA levels by reverse transcription plus polymerase chain reaction amplification (RT-PCR). Our synthetic EGF mRNA construct contains the same flanking primer sequences used to amplify mature EGF message, but 70 bases were eliminated from the center of the 277-base EGF sequence to permit the PCR product of this internal standard to be distinguished by its smaller size (EGF 207). This synthetic mRNA also contains a poly(A)tail, which permits it to be reverse transcribed. We then added a range of concentrations of this internal standard mRNA to aliquots of total RNA from each pair of thyroid lobes and determined the concentration of EGF 207 at which the PCR primers were incorporated equally into the 277 and 207 bands after RT-PCR. Thyroid RNA from male Balb/c mice treated with T4 (0.25 micrograms/g.day) for 14 days contained 2.8-fold more EGF mRNA than RNA from control mice (P < 0.01). Competitive RT-PCR EGF mRNA levels were determined for thyroid RNA samples from mice treated with T4 for various times up to 14 days. The most significant increase occurred after 1 day's treatment (P < 0.005). This demonstration of a thyroid hormone-dependent increase in the level of thyroidal EGF mRNA adds support to the concept that EGF may function as an autocrine/paracrine regulator of thyroid function.

The specific interactions of HMG 1 and 2 with negatively supercoiled DNA are modulated by their acidic C-terminal domains and involve cysteine residues in their HMG 1/2 boxes.

Sedimentation and gel retardation studies show a stronger interaction of HMG 1 and 2 with negatively supercoiled DNA than with linear, nicked-circular, or positively supercoiled ds-DNA. An apparent unwinding angle of 58 degrees was obtained for HMG 1 and 2 when assayed by protection of negatively supercoiled DNA from topoisomerase I relaxation or when assayed by the supercoiling of nicked-circular DNA with T4 DNA ligase. The protection of negatively supercoiled DNA was linear up to molar ratios of about 250:1. There was little change in binding reactions or in the protection of supercoiled DNA at ratios above 250:1, indicating that both activities saturate and that HMG 1 and 2 have binding site sizes of about 20 bp. P1, the major tryptic fragment of HMG 1 or 2 which retains the two DNA binding HMG 1/2 boxes, displays a 2-fold increase in binding to all types of ds-DNA compared to intact HMG 1 or 2. However P1 protects negatively supercoiled DNA from topoisomerase I relaxation about 5-fold less than intact HMG 1 or 2. Complete protection with P1 occurs at a molar ratio 1040:1, indicating a DNA binding site size of about 4 bp and an apparent unwinding angle of 10 degrees. P1 binding to closed-circular ss-DNA also involves a binding site of about 4 bp. Adding the acidic C-terminal fragment to P1 reversed its binding and allowed topoisomerase I to relax supercoiled DNA. These findings highlight the importance of the acidic C-terminal domains of HMG 1 and 2 in limiting electrostatic interactions of the HMG 1/2 boxes with ds- or ss-DNA. N-Ethylmaleimide inhibited the binding of intact HMG 1 or 2 to negatively supercoiled DNA, but did not inhibit the electrostatic binding of HMG 1 or 2 to ss-DNA, or of P1 to any form of DNA (ds or ss). These results suggest that cysteine residues are involved in the specific interaction of HMG 1 or 2 with negatively supercoiled DNA and that the acidic C-terminal domains modulate an intramolecular conformational change involving sulfhydryls within the HMG 1/2 boxes.

Studies on the biological activity of triiodothyronine sulfate.

Hepatic microsomes and isolated hepatocytes in short term culture desulfate T3 sulfate (T3SO4). We, therefore, wished to determine whether T3SO4 could mimic the action of thyroid hormone in vitro. T3SO4 had no thyromimetic effect on the activity of Ca(2+)-ATPase in human erythrocyte membranes at doses up to 10,000 times the maximally effective dose of T3 (10(-10) mol/L). In GH4C1 pituitary cells, T3SO4 failed to displace [125I]T3 from nuclear receptors in intact cells or soluble preparations. Thus, T3SO4 was not directly thyromimetic in either an isolated human membrane system or a pituitary cell system in which nuclear receptor occupancy correlates with GH synthesis. Thyroid hormones inhibit [3H]glycosaminoglycan synthesis by cultured human dermal fibroblasts, and T3SO4 displayed about 0.5% the activity of T3 at 72 h. Human fibroblasts contained roughly the same level of microsomal p-nitrophenyl sulfatase activity as that previously observed in hepatic microsomes. Propylthiouracil (50 mumol/L) did not affect the action of T3SO4, suggesting that deiodination was not important for this activity of T3SO4. Thus, it appears T3SO4 has no intrinsic biological activity, but, under certain circumstances, may be reactivated by desulfation.

Thyroxine increases epidermal growth factor levels in the mouse thyroid in vivo.

Immunoreactive epidermal growth factor (EGF) has been detected in the thyroid, which raises questions concerning the source of thyroidal EGF and what affects its levels. We therefore have examined the effects of manipulating thyroid function on the immunoreactive EGF levels in plasma, thyroid, submaxillary gland (SMG), and kidney of adult male BALB/c mice, and we also analyzed the prepro-EGF messenger RNA in these tissues. Groups of six mice received daily injection of T4 (1 microgram, 5 micrograms, or 25 micrograms) or bovine TSH (1 mU) for up to 14 days. The plasma EGF concentration was 0.31 +/- 0.01 ng/ml in control animals, and T4 (5 micrograms) decreased the plasma levels by about one-half within 1 week. The thyroidal EGF was 0.50 +/- 0.14 ng/mg protein in control animals, and T4 (5 micrograms) increased the thyroidal EGF 8-fold within 1 week. There was a negative correlation between plasma and thyroidal EGF concentration (r = -0.93, P less than 0.01). Increasing doses of T4 also increased the SMG EGF content, while plasma levels fell (r = -0.88, P less than 0.01). The TSH treatment did not significantly alter the plasma or tissue EGF levels. Studies on mRNA prepared from these three tissues, using the reverse transcription and the polymerase chain reaction, indicated that the prepro-EGF mRNA was present in thyroid, SMG, and kidney. In conclusion, it appears that at least some thyroidal EGF is synthesized in the thyroid. Our observations that T4 increases the level of intrathyroidal EGF in a dose- and time-dependent fashion, while plasma levels fall, suggest the possibility that intrathyroidal EGF represents a shortloop feedback for the autocrine and/or paracrine regulation of thyroid function.

Cyclic adenosine 3',5'-monophosphate-dependent phosphorylation of HMG 14 inhibits its interactions with nucleosomes.

The high mobility group protein HMG 14, which is preferentially associated with nucleosomes containing active gene sequences, is phosphorylated on different sites according to the tissue and stimulus being studied. In the thyroid, HMG 14 displays TSH-dependent phosphorylation that is mediated by cAMP-dependent protein kinase (A-kinase). We have, therefore, studied how phosphorylation of HMG 14 on its major and minor A-kinase sites (Ser-6 and -24) affects its interactions with nucleosomes and various forms of DNA, since this could reflect a means of regulating its function of binding to active chromatin. Approximately twice as much Ser-6 phospho- and 4 times as much Ser-6,24 diphospho-HMG 14 were required to produce the same degree of nucleosome band displacement as that caused by native unphosphorylated HMG 14. Phosphorylation also reduced the ability of HMG 14 to protect the ends of nucleosomal DNA from thermal denaturation. When the electrophoretic mobility of naked DNA was examined, the Ser-6 phospho-HMG 14 was about half as effective as native HMG 14 in retarding the various forms of double stranded DNA, and Ser-6,24 diphospho-HMG 14 was even less effective. Our data demonstrate that electrostatic interactions between DNA and basic amino acids in two highly conserved regions (residues 1-5 and 16-27) can be modulated by phosphorylation at Ser-6 and Ser-24. The ability of mammalian HMG 14, but not HMG 17, to display hormone-dependent phosphorylation may indicate a route for differentially modulating their binding to transcriptionally active chromatin.

HMG 14 and protamine enhance ligation of linear DNA to form linear multimers: phosphorylation of HMG 14 at Ser 20 specifically inhibits intermolecular DNA ligation.

HMG 14 and protamine can be used to enhance intermolecular ligation of low concentrations of linear DNA. Adding HMG 14 (50 moles per mole DNA) caused 50% of blunt-ended DNA to form predominantly dimers, and all cohesive-ended DNA to form multimers (greater than 6-mer) in response to T4 ligase. Protamine was maximally effective at 40:1, producing mostly dimers and trimers. Adding higher concentrations of HMG 14 did not affect the ligation pattern of cohesive-ended DNA, while higher concentrations of protamine inhibit the formation of multimers. Phosphorylation of HMG 14 at Ser 20 by Ca(++)-phospholipid dependent protein kinase abolished the ability of HMG 14 to stimulate intermolecular ligation, but did not substantially interfere with intramolecular ligation, or the binding of HMG 14 to linear or circular DNA as assessed by gel mobility. Thus Ser 20, which is located in the amino terminal DNA-binding domain of HMG 14, appears to modulate DNA-DNA interactions.

Epidermal growth factor inhibits radioiodine uptake but stimulates deoxyribonucleic acid synthesis in newborn rat thyroids grown in nude mice.

We have studied the effect of altering the level of circulating epidermal growth factor (EGF) on the function and growth of newborn rat thyroids transplanted into nude mice. Preliminary studies confirmed that sialoadenectomy reduced circulating EGF levels in nude mice (from 0.17 +/- 0.02 to 0.09 +/- 0.02 ng/ml), and that ip injection of 5 micrograms EGF raised EGF levels (the peak level of 91.7 +/- 3.3 ng/ml was achieved at 30 min, with a subsequent half-life of about 1 h). The radioiodine uptake by newborn rat thyroid transplants in the sialoadenectomized and sham-operated animals correlated inversely with the circulating EGF levels determined when the mice were killed (r = -0.99). Low-dose TSH treatment (0.1 microU/day) generally stimulated the radioiodine uptake, but high-dose TSH groups (100 microU/day) were not significantly different from the control group. The 5-day nuclear [3H]thymidine labeling index was 6.8 +/- 0.5% IN newborn rat thyroid transplants grown in sialoadenectomized animals, 13.1 +/- 0.3% in sham-operated animals, and 16.8 +/- 0.5% in nude mice receiving 5 micrograms EGF ip daily. In general, both low-dose and high-dose TSH promoted DNA synthesis under low EGF conditions but were ineffective in the presence of higher levels of EGF. Adult rat thyroid transplants showed no significant responses. Although sialoadenectomy may alter other factors besides EGF, it appears that changes in the levels of circulating EGF within the physiological range affect the function and growth of newborn rat thyroid transplants. Circulating EGF may play a role in thyroid maturation and may also be involved in the regulation of thyroid function throughout life.

High mobility group protein 1 preferentially conserves torsion in negatively supercoiled DNA.

HMG 1 is known to bind to a variety of DNAs and to unwind nicked and closed circular DNA. We now report evidence that it has a significantly higher unwinding angle on negatively supercoiled DNA than on the other torsional forms. The degree of unwinding observed on nicked circular DNA depends on the purity of the HMG 1 preparation used. HMG 1 from CM-Sephadex has an unwinding angle of 28.8 degrees, compared to 7.2 degrees for the purer preparation obtained from Mono S, suggesting that contaminating strand-separating activity is removed by the additional purification step. The subsequent studies on closed circular forms of DNA were all performed using the purer HMG 1. After preincubation of highly negatively supercoiled DNA (sigma = -0.040) with HMG 1, the DNA-protein mixture was relaxed with Escherichia coli topoisomerase I. At molar ratios of less than 100:1 (HMG 1 to DNA), negatively supercoiled DNA displays a dose-dependent change in the linking number, indicating an unwinding angle of 57.6 degrees. HMG 1 protects 50% of highly negatively supercoiled DNA from E. coli topoisomerase I at a molar ratio of 100:1, and protects all supercoils at a molar ratio of 200:1, indicating saturation of the DNA at this concentration. HMG 1 also protects highly negatively supercoiled DNA from calf thymus topoisomerase I, with an apparent unwinding angle of 57.6 degrees. Moderately negatively supercoiled DNA (sigma = -0.018), but not moderately positively supercoiled DNA (sigma = +0.011), competes for the protective effect of HMG 1 on highly negatively supercoiled DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

A one-step preparative method for separating SER 6-phosphorylated HMG 14 from unphosphorylated HMG 14 and in vitro phosphorylation reaction components.

While clear evidence exists for the regulation of the phosphorylation of the very basic high mobility group (HMG) and histone chromatin proteins, the physiological role of their phosphorylation remains poorly understood. Elucidation of these roles has been difficult, in part, because of the inability to obtain sufficient quantities of purified phosphorylated derivatives. We have used Mono S cation-exchange chromatography to prepare milligram quantities of pure Ser 6-phosphorylated HMG 14 (Ser 6-PO4-HMG) from unphosphorylated Mono S-purified calf thymus HMG 14 following in vitro phosphorylation with cAMP-dependent protein kinase (A-kinase). In one step, this technique separates the phosphorylated derivative from A-kinase, ATP, unphosphorylated HMG 14, and a minor phosphorylated by-product which evidence suggests may be the previously reported Ser 6, 24-diphospho-HMG 14. Mono S chromatography also enhances the purity of calf thymus HMG 14 prepared by perchloric acid extraction, acetone and ethanol precipitations, and CM-Sephadex chromatography. In addition, it permits the detection of apparent microheterogenous forms of both unphosphorylated and Ser 6-PO4-HMG 14. The significant reductions in binding affinity resulting from the incorporation of phosphate groups into HMG 14 suggest that Mono S chromatography could have more general application in the isolation of phosphorylated derivatives of other basic proteins, including other chromatin-associated DNA-binding proteins which are known to undergo specific phosphorylation. It would especially be useful when the proteins and their phosphorylated derivatives bind more tightly to Mono S than the kinases used for their phosphorylation.