Effect of pioglitazone on survival and renal function in a mouse model of polycystic kidney disease.

BACKGROUND/AIMS: Cystic epithelia in polycystic kidney disease display features similar to malignant cells. Thiazolidinediones have been shown to have anti-neoplastic properties, therefore we tested the hypothesis that pioglitazone reduces cyst formation, improves renal function, and prolongs survival in a mouse model of polycystic kidney disease. METHODS: PC-Pkd1-KO mice, which have homozygous mutations of the Pkd1 gene in principal cells, were used. On the day after giving birth, mothers were fed standard mouse chow with or without pioglitazone (30 mg/kg chow). After weaning, the assigned diet was continued. At 1 month of age, blood pressure was measured and animals were sacrificed to determine kidney weight, body weight, and serum urea. Kidneys were evaluated for proliferation using Ki-67, apoptosis using TUNEL analysis, and cyst number using MRI. Survival was observed. RESULTS: Pioglitazone did not alter renal function, cell proliferation, apoptosis, or cyst formation in animals with polycystic kidney disease, however it did increase survival. Pioglitazone reduced blood pressure in PC-Pkd1-KO, but not in controls. CONCLUSION: These findings suggest that pioglitazone may have a unique antihypertensive effect in polycystic kidney disease, and that such an effect may promote improved survival.

Identification of a p53 response element in the promoter region of the hMSH2 gene required for expression in A2780 ovarian cancer cells.

Defects in the human MSH2 mismatch repair system have been implicated in cellular mutagenesis, tumorigenesis, and chemotherapeutic resistance. The current studies characterized the 5' upstream proximal promoter region of the hMSH2 gene using transient transfection of A2780 ovarian cancer cells. Serial deletions of a 1.88-kb fragment of the proximal promoter region of the hMSH2 gene revealed that promoter activity was restricted to the first -281 bp. Targeted deletions within this -281 bp region coupled with specific sequence mutagenesis identified a response element for the p53 tumor suppressor protein located between -242 and -222 bp. The -242 hMSH2 p53 element is configured as a direct tandem repeat palindrome with 80% homology to the p53 consensus binding sequence. Co-transfection of an hMSH2 reporter and p53 expression vector into the p53-null cell line SK-OV-3 produced 10-fold enhanced transcription, which was lost when the -242 to -222 p53 binding site was mutated. These results clearly demonstrate the presence of a previously unidentified p53 response element in the hMSH2 proximal promoter. Its location at -242 bp upstream of the start site of transcription is distinct from two previously reported p53 sites at -447 and -416, which transactivate in Saos-2 cells (Scherer, S. J., Maier, S. M., Seifert, M., Hanselmann, R. G., Zang, K. D., Muller-Hermelink, H. K., Angel, P., Welter, C., and Schartl, M. (2000) J. Biol. Chem. 275, 37469-37473). Finally, in sharp contrast to their activity in Saos-2 cells, deletion of the -447 and -416 sites in A2780 cells had no effect on hMSH2 promoter activity. Thus, it appears that p53 regulates hMSH2 expression through multiple cell type-specific DNA response elements.

Purkinje cell protein-2 cis-elements mediate repression of T3-dependent transcriptional activation.

Previous studies in our laboratory show that triiodothyronine upregulates expression of the cerebellar Purkinje cell-specific gene Pcp-2 during the first 2 weeks of rat neonatal life. A specific thyroid hormone response element, the A1 TRE, mediates this regulation. The finding that the contiguous 68 bases (-267/ -199) of the Pcp-2 promoter 3' to the A1 TRE repressed T3 response in transactivation studies suggested that this sequence could play a role in preventing premature T3-dependent activation of Pcp-2 in the fetus. We now show that deletion of this region resulted in enhanced T3-dependent activation of the native Pcp-2 promoter. The sequence is not a generalized silencer since it does not alter basal activity of mouse mammary tumor virus (MMTV) or thymidine kinase (TK) promoters. Deletion and linker scanning studies indicate that the 5' 30 bases of the -267/ -199 region mediate most of the response silencing activity. The -267/ -199 region also attenuates T3-induced transactivation mediated by other TREs. Gel shift analysis reveals that nuclear proteins from fetal but not adult brains complex with the -267/ -199 region, supporting the hypothesis that this region binds proteins that suppress Pcp-2 expression early in brain development.

Transient stimulation of myelin basic protein gene expression in differentiating cultured oligodendrocytes: a model for 3,5,3'-triiodothyronine-induced brain development.

We compared the regulation of myelin basic protein (MBP) gene expression by T3 in differentiating oligodendrocytes in culture with that previously observed by us in the neonatal rat brain. As in intact brain, expression of the T3R alpha gene preceded that of the T3R beta gene. Although the absence of T3 retarded the rate of accumulation of MBP messenger RNA, the level ultimately attained was similar to that reached in the presence of T3. This relationship mirrored the pattern observed in the neonatal brain. Transient transfection experiments showed that T3 regulates MBP expression at the transcriptional level, but only for a limited period during differentiation. These observations imply that the early rise of MBP messenger RNA is T3 dependent, whereas the terminal levels are maintained independently of T3. Both the T3-dependent and, surprisingly, the T3-independent expression of MBP require the presence of an intact T3 response element. T3 receptor may regulate MBP expression in a ligand-independent manner, or a nuclear factor other than T3 receptor may bind to the T3 response element of MBP to regulate terminal gene expression. These findings support the use of differentiating oligodendrocytes as a model of T3-induced brain development.

Thyroid hormone receptor isoform content in cultured type 1 and type 2 astrocytes.

Immunohistochemical studies previously reported from this laboratory showed that astrocytes in adult rat brain appear devoid of all thyroid hormone receptor (TR) isoforms. These findings, however, contrast with reports of measurable nuclear T3 binding in astrocytes in cell culture. To address this discrepancy, TR protein and messenger RNA (mRNA) content of type 1 and type 2 astrocytes in culture were assayed. Type 1 cells represent astrocytes present in brain in vivo. Type 2 astrocytes differentiate in culture from bipotential progenitor O-2A cells in the presence of serum. Under serum-free conditions, these progenitor cells differentiate into oligodendroglia. Total nuclear T3 binding capacity in both type 1 and type 2 astrocytes was approximately 3000 sites/cell. Northern blots showed the presence of mRNA for TRbeta1, TRalpha1, and TRalpha2 in type 2 cells but failed to reveal the presence of these mRNAs in type 1 astrocytes. Moreover, Northern blots also failed to reveal TRbeta2 mRNA in both type 1 and type 2 astrocytes. These findings, therefore, raised a question as to which receptor isoform was responsible for the nuclear binding capacity observed in type 1 astrocytes. As anticipated, immunocytochemical analysis demonstrated prominent nuclear signals for TRbeta1, TRalpha1, and TRalpha2 mRNA in type 2 astrocytes but failed to demonstrate TRbeta1, TRalpha1, or TRalpha2 in type 2 astrocytes. Application of RT-PCR, however, revealed the presence of low levels of TRbeta2 mRNA in type 1 astrocytes. When stained with a specific anti-TRbeta2 antiserum, both type 1 and type 2 astrocytes showed a strong fluorescent signal concentrated in the nucleus. These data indicate that under the special conditions of cell culture, expression of the TRbeta2 isoform in type 1 accounts for the measured nuclear T3 binding capacity.

A Purkinje cell protein-2 intronic thyroid hormone response element binds developmentally regulated thyroid hormone receptor-nuclear protein complexes.

Two thyroid hormone response elements (TREs), designated A1 TRE (-295/-268) and B1 TRE (+207/+227), have been identified within the Purkinje cell-expressed Pcp-2 gene. Previous studies have characterized the A1 TRE (Zou et al., 1994). This article analyzes the structural and functional characteristics of the intronic B1 TRE. The B1 sequence contains four overlapping TRE half-sites. The 3' DR4 motif, consisting of the second and forth half-sites, is responsible for the T3 induction observed with the B1 sequence. Gel-shift analysis reveals developmentally regulated complexes that are abundant in the fetus and at birth and then fall precipitously in the neonate bind to B1. The observed time-course of these complexes varies inversely with the rise in Pcp-2 expression, thus raising the possibility that the complexes may represent inhibitory factors. Supershift analysis indicates that endogenous TR alpha 1 is present in the fetal nuclear protein complexes that bind to B1. Competition analysis also indicates the second B1 TRE half-site is important in binding the TR alpha 1-TRAP complexes. These studies suggest that the B1 sequence may bind potential TR alpha 1-TRAP repressor complexes in the fetus, whereas in the neonate, these TRE sites may be involved in the activation of Pcp-2 by binding other TR-TRAP-activating complexes.

Plasticity in the synthesis and storage of substance P and calcitonin gene-related peptide in primary afferent neurons during peripheral inflammation.

Several indices of peptidergic, primary afferent neural transmission in rat at the level of the lumbar spinal cord exhibited differential changes over time in response to adjuvant-induced inflammation of the hindpaw. The indices were measurements of the production of messenger RNA encoding the precursors for substance P and calcitonin gene-related peptide in dorsal root ganglia, the storage of substance P and calcitonin gene-related peptide in the dorsal spinal cord and the release of the peptides evoked by application of capsaicin to the dorsal spinal cord. A 47% decrease in the content of immunoreactive substance P in the dorsal half of the lumbar spinal cord, as determined by radioimmunoassay, was measured at 6 h following the injection of complete Freund's adjuvant into the hindpaw. Decreased content of immunoreactive SP persisted for four days, but was no longer present at eight days after the adjuvant injection. The content of immunoreactive calcitonin gene-related peptide in the dorsal spinal cord was decreased by 29% at one day following the injection of adjuvant into the rat hindpaw and 43% at two days; the content then increased to a level greater than that of control animals at eight days. The amount of messenger RNA encoding preprotachykinin and prepro-calcitonin gene-related peptide in L4-L6 dorsal root ganglia was determined from northern blot analysis of the total messenger RNA extracted from the dorsal root ganglia. Each species of messenger RNA had increased compared to the control animals at two days following the injection of adjuvant into the rat hindpaws and remained elevated after eight days. Thus, an increase in the messenger RNAs encoding substance P and calcitonin gene-related peptide in the dorsal root ganglia preceeded the recovery of the content of the peptides in the spinal cord. Morphometric studies of calcitonin gene-related peptide-immunoreactive perikarya in the L4 dorsal root ganglia indicated that the increase in messenger RNA occurred in neurons of the size that normally express calcitonin gene-related protein. Radioimmunoassay of the superfusate of the dorsal half of the lumbar spinal cord was used to measure the release of immunoreactive substance P and immunoreactive calcitonin gene-related protein in vitro. Although the basal release of immunoreactive substance P and immunoreactive calcitonin-gene related protein from the dorsal spinal cord was constant throughout the time points examined, changes occurred in the release of peptide evoked by 10 microM capsaicin. The capsaicin-evoked release of immunoreactive substance P was decreased at 6 h and eight days post-injection of adjuvant.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunofluorescent localization of thyroid hormone receptor isoforms in glial cells of rat brain.

The three currently recognized T3 binding thyroid hormone receptor (TR) isoforms, TR alpha 1, TR beta 1, and TR beta 2, arise from two distinct genes (alpha and beta), whereas two closely related non-T3-binding receptor variants, collectively designated TR alpha 2, arise from alternate splicing of the alpha gene transcript. Using a panel of specific antisera to these isoforms we have assessed the presence or absence of TRs in oligodendrocytes and astrocytes of rat cerebrum and cerebellum. Inferences as to colocalization of the receptor isoforms and cell-specific marker proteins were based on immunohistochemical analysis of the differential emissions of paired immunofluorescent probes. Antisera against myelin basic protein (MBP) identified oligodendroglia, and glial fibrillary acidic protein identified astrocytes. MBP-positive oligodendrocytes displayed positive fluorescent signals with each of the three TR isoform-specific antisera and the antiserum to the receptor variants. These findings are consistent with the concept that the MBP gene is a direct target for thyroid hormone action. TR immunoreactivity appeared to localize primarily to the nuclei of these cells. In contrast, we observed no immunofluorescent signals for any of the TR isoforms in glial fibrillary acidic protein-positive astrocytes. These findings raise the possibility that any effect of thyroid hormone on astrocyte function and structure is mediated indirectly as a result of interaction of thyroid hormone with receptors situated in nonastrocyte cells or as a result of nonnuclear mechanisms.

Identification of thyroid hormone response elements in rodent Pcp-2, a developmentally regulated gene of cerebellar Purkinje cells.

In a previous study, we have shown that in vivo expression of the cerebellar Purkinje cell-specific gene Pcp-2 is regulated by thyroid hormone (T3) during neonatal development. In addition, transient cotransfection studies using thyroid hormone receptors (TRs) and a Pcp-2-lacZ construct pointed to direct regulation of Pcp-2 gene expression by T3. Therefore, we have initiated the following series of studies to define more precisely the location of the thyroid hormone regulatory elements in the Pcp-2 gene. By transfection and in vitro receptor binding analyses, we have identified two thyroid hormone response elements, A1 (-295/-268) and B1 (+207/+227). A1 contains a central half-site flanked by two similar half-sites. B1 contains two pairs of alternate half-sites. When these elements were ligated to the modified mouse mammary tumor virus promoter (delta MMTV), both induced a 8-14-fold expression of the reporter gene, but only in the presence of T3. Gel mobility assays demonstrated that both A1 and B1 bind TRs in the presence of thyroid hormone receptor auxiliary proteins or the retinoid X beta receptor. Mutations of the G residues to T within the individual half-site sequences of A1 caused a variable decrease in the transactivation of the MMTV-CAT construct and a corresponding reduction in TR binding in vitro. Thus, mutational analysis of A1 pointed to the interaction of the flanking half-site motifs with the central AGGTCA half-site. Interestingly, lengthening of the A1 sequence at its 3'-end caused a progressive dampening of the T3 response. The results suggest that the neighboring sequence may function as a silencer of the A1 element. Since thyroid hormone regulation of Pcp-2 is manifest only during the first 2 weeks after birth, we hypothesize that A1 and B1 act as T3-dependent response elements operative only during early neonatal Purkinje cell development and that their function is suppressed by a neighboring silencer element operative when expression of Pcp-2 becomes hormone-independent.

Molecular mechanisms of thyroid hormone action. A physiologic perspective.

At present, it appears abundantly clear that thyroid hormone exerts its major action at the nuclear level by regulating the level of mRNAs of specific genes. There are at least three TR isoforms that mediate hormonal effects at the tissue level. Characterization of the functional domains of these receptor isoforms is as yet incomplete, and the possibility that these receptors could have ligand-independent functions is a matter under current investigation. TRs are now recognized as members of a large superfamily of transactivating proteins involved in the regulation of gene expression. Recent studies have shown an unexpected degree of complexity in the nature of the association of the T3 receptors and the DNA of target genes. They have vividly pointed out the multiple interactions possible between the T3-receptor complex and other proteins participating in the process of gene regulation. These insights have provided a solid base for understanding differences in the gradation of thyroid hormone effect from one tissue to another. The microdissection of the molecular process that has occurred in the past 20 years has proceeded in part through the application of relatively artificial in vitro systems and assays. Whereas such approaches have undoubtedly reaped rich rewards in pointing out potential or possible mechanisms, they do not define the actual workings in the animal. Additional studies designed to examine at the molecular level the operation in vivo of physiologic networks influenced by thyroid hormones appear as an essential next step in understanding the biology of the hormone system. The application of transgenic models should materially assist such efforts.

Immunocytochemical delineation of thyroid hormone receptor beta 2-like immunoreactivity in the rat central nervous system.

The thyroid hormone receptors (TR) are nuclear proteins that include TR alpha and TR beta subtypes, each encoded by a separate gene. Both TR alpha and TR beta give rise to several isoforms of which three, TR alpha 1, TR beta 1, and TR beta 2 bind T3 and mediate the action of thyroid hormone. Although TR beta 2 was initially thought to be confined to the anterior pituitary, we recently observed small quantities of TR beta 2 messenger RNA (mRNA) by polymerase chain reaction analysis of discrete hypothalamic regions. To further examine the distribution of TR beta 2 in the brain, we performed immunocytochemical studies using a highly specific antiserum to TR beta 2, raised against a unique amino acid sequence (TR beta 2[131-145]) that is not present in the other known TRs. This antiserum immunoprecipitated TR beta 2 but not TR alpha 1 or TR beta 1. Immunoreactive TR beta 2 was widely distributed throughout the brain and primarily localized to the cell nucleus. Particularly intense immunostaining was present in the cerebral cortex, cerebellum, and hypothalamus, including regions where TR beta 2 mRNA had not previously been identified. In addition, immunoprecipitation of nuclear extracts with anti-TR beta 2 reduced total T3 binding capacity by approximately 20%, suggesting that immunoreactive TR beta 2 comprises a substantial portion of the total content of nuclear thyroid hormone binding proteins. These studies demonstrate that immunoreactive TR beta 2 is more widely represented in the central nervous system than previously suspected and may play an important role in mediating the action of T3 in many different regions of the brain. The finding of TR beta 2-like material could be due to a disproportionately high ratio of the TR beta 2 translation product and its mRNA in certain regions of the brain, or could indicate the existence of a novel TR beta 2-related protein that is important for T3 binding.

Ontogeny of hepatic nuclear triiodothyronine receptor isoforms in the rat.

We have determined the contribution of the thyroid hormone receptor (TR) isoforms TR alpha 1 and TR beta 1 to the postnatal rise in rat hepatic nuclear T3-binding capacity. In agreement with previous studies, total hepatic nuclear binding capacity rose by about 8-fold from the 19th day of gestation to young adulthood at 2 months of age (0.10 +/- 0.03 to 0.86 +/- 0.17 pmol/mg DNA). The levels of specific TR species were measured by immunoprecipitation of T3-binding activity from hepatic extracts using a panel of antisera directed against specific regions of the TR isoforms. The difference between receptor immunoprecipitated with antibody against TR beta 1 and that precipitated with an antibody against an identical region in both TR beta 1 and TR alpha 1 was tentatively assumed to represent TR alpha 1. TR alpha 1 accounted for virtually all T3-binding activity in fetal liver on gestational day 19 (G19), increased by 2-fold shortly after birth, and remained constant thereafter. TR alpha 1 mRNA, on the other hand, was highest in concentration on G16 and fell by 50-75% in the adult. TR beta 1 was undetectable by immunoprecipitation of hepatic extracts from fetuses on G19. However, Northern analysis showed the presence of TR beta 1 mRNA in the fetal liver, which rose in concentration by 3- to 4-fold in late gestation and then remained constant. The contribution of TR beta 1 to total binding capacity rose to 33% and 40% on postnatal days 15 and 30, respectively, and to 80% in the adult liver. Immunohistochemical analyses of hepatic sections confirmed the presence of very low levels of TR beta 1 in fetal liver as early as G16 and G19, and a sharp rise in TR beta 1 protein concentration in the postnatal period. This indicated that the increase in TR beta 1-binding capacity results from increased TR beta 1 mass. The increase in TR beta 1-binding capacity, thus, is due to increased translational efficiency of the beta 1 mRNA or stabilization of the TR beta 1 protein. The prominence of TR alpha 1 in both rat fetal liver and fetal brain, as previously demonstrated in our laboratory, raises the possibility that this receptor isoform may carry out specialized functions in the fetus and that TR beta 1 subserves still other functions at later stages of development.(ABSTRACT TRUNCATED AT 400 WORDS)

Beta 1 isoform-specific regulation of a triiodothyronine-induced gene during cerebellar development.

Although tissue-specific expression of the alpha 1 and beta 1 thyroid hormone receptors (TR-alpha 1 and TR-beta 1) suggests isoform-specific function, transfection studies to date have failed to show consistent differences in their ability to regulate gene expression. We here provide evidence that TR-beta 1 but not TR-alpha 1 regulates the expression of the gene coding for PCP-2 in cerebellar Purkinje cells during neonatal rat development and that such regulation appears to be both T3 dependent and T3 independent. Examination of neonatal rats revealed that the levels of three mRNAs expressed in cerebellar Purkinje cells (myoinositol-1,4,5-triphosphate receptor, calbindin, and PCP-2) rise from neonatal day 1 to day 15. This rise is preceded by the previously documented surge in brain T3 and TR-beta 1. Methimazole-induced hypothyroidism sharply reduces, but does not abolish, the rise in these mRNAs. Concomitant T3 administration normalizes the process. In order to establish more directly the role of TR-beta 1 and T3, cotransfection experiments were performed in CHO cells with PCP-2-lacZ construct and TR isoforms. These studies showed that TR-beta 1, even in the absence of T3, regulated the expression of the transfected PCP-2 construct. T3 augments the response to TR-beta 1 alone by 40% (P < .01). TR-alpha 1 had no effect on PCP-2-lacZ expression either in the presence or absence of T3.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of thyroid hormone status on the expression of the mRNAs of components of the lipolytic regulatory cascade in brown adipose tissue.

1. The levels of mRNAs for RII beta and G beta were about 50% lower in brown adipose tissue (BAT) from hyperthyroid than from hypothyroid rats. 2. Treatment of hypothyroid rats with T3 resulted in a 50% decrease in mRNAs for RII beta and G beta in BAT occurring by 12 hr after treatment. 3. The levels of mRNAs for hormone-sensitive lipase, G alpha s and C alpha in BAT were unchanged by thyroid hormone status. 4. The results suggest that thyroid hormone may be involved in negative regulation of the expression of RII beta and G beta at the transcriptional level in BAT.

Quantitation of rat tissue thyroid hormone binding receptor isoforms by immunoprecipitation of nuclear triiodothyronine binding capacity.

A panel of anti-thyroid hormone receptor (TR) antisera were generated to allow direct assay of the concentrations of the alpha 1 and beta 1 receptor isoforms in nuclear extracts from adult rat liver, kidney, brain and heart, and fetal brain. An antiserum, immunoglobulin G (IgG)-beta 1, raised against amino acid sequence 62-92 of the rat TR-beta 1 specifically precipitated only TR-beta 1 in vitro translation products. A second antiserum, IgG-alpha 1/beta, generated against a sequence that is identical in the ligand binding region of rat TR-alpha 1 and TR-beta isoforms immunoprecipitated both TR-alpha 1 and -beta 1 translation products. These IgG preparations were used to specifically immunoprecipitate thyroid hormone receptor binding activity from nuclear extracts. IgG-beta 1 cleared almost 80%, and the IgG-alpha 1/beta immunoprecipitated nearly all binding from hepatic nuclear extracts. This distribution of TR protein, 80% beta 1 and 20% alpha 1, is the same as previously reported for their respective mRNAs in liver. In heart, kidney, and brain IgG-beta 1 cleared 45, 43, and 28% of total binding, respectively, and IgG-alpha 1/beta cleared all T3 binding activity from these tissues. In agreement with an earlier study, marked variations in specific protein/mRNA ratios were noted among these tissues. Consistent with our earlier report of the presence of only very low levels of TR-beta 1 mRNA in fetal brain, IgG-beta 1 cleared just 5% of binding in this tissue. Studies using an antiserum (IgG-ch) generated against homologous segments of the hinge region in both TR-alpha 1 and -beta 1 yielded results which contrasted sharply with those of IgG-alpha 1/beta. Whereas IgG-ch could also immunoprecipitate virtually all binding from hepatic extracts it cleared only 40-50% of binding from the other tissues, including fetal brain in which TR-alpha 1 accounts for greater than 90% of binding protein. The data suggest the presence of posttranslational modification of the TR-alpha 1 protein in the hinge region, consistent with the presence in this segment of potential phosphorylation sites.

Prolonged fasting reduces rat hepatic beta 1 thyroid hormone receptor protein without changing the level of its messenger ribonucleic acid.

The level of hepatic nuclear T3-binding capacity falls in rats subjected to fasting. To define the mechanism underlying these changes, we have assayed in liver the concentration of the mRNA coding for the beta 1-receptor (beta 1-TR) isoform, the total nuclear T3-binding capacity, and the fraction of the total binding capacity that can be specifically immunoprecipitated with an anti-beta 1-TR immunoglobulin G preparation. Although no changes in beta 1-TR mRNA concentration were noted, we observed a 60% fall in total binding capacity. beta 1-TR mRNA levels were preserved despite a 50% fall in total poly(A)+ RNA. The fall in beta 1-TR protein, however, was consistent with a generalized decrease in total hepatic protein content. This study provides yet another instance in which measurement of receptor mRNA is not consonant with the behavior of the nuclear T3 receptor protein.

Immunofluorescence localization of thyroid hormone receptor protein beta 1 and variant alpha 2 in selected tissues: cerebellar Purkinje cells as a model for beta 1 receptor-mediated developmental effects of thyroid hormone in brain.

Rat c-erbA beta 1 mRNA rises in cerebrum during the first 10 days of life, coincident with an increase in tissue triiodothyronine (T3) levels and T3-dependent brain development. These data suggest that the beta 1 receptor may mediate the T3 effect. However, in cerebellum c-erbA beta 1 mRNA levels were very low. Since cerebellar development, including dendritic arborization of Purkinje cells, is a T3-sensitive process, we assessed the levels of the beta 1 receptor protein in cerebellum during development. Antisera to unique peptide regions of beta 1 were raised. Their specificity was demonstrated by specific immunoprecipitation of the in vitro translated product, 85% immunoprecipitation of the T3 binding activity in hepatic nuclear extracts, and Western blot analysis of tissue extracts. Immunohistochemical studies using anti-beta 1 antiserum stained liver nuclei but not testis nuclei, which contain no T3 binding activity or beta 1 mRNA. In cerebellar Purkinje cells, an immunofluorescent signal, localized to the nucleus and more intense than that seen in the liver, was observed. A positive but weaker signal was also present in the granule cells. Thus, we may infer that the cerebellum contains significant concentrations of beta 1 receptor protein despite the low beta 1 mRNA content. Both the intensity of staining in Purkinje cell nuclei and immunoprecipitable beta 1 receptor binding capacity rose in the neonatal period. Antiserum to the non-T3 binding alpha 2 variant protein was also prepared and a distinctive pattern of fluorescence was observed. Strong fluorescence was seen in the nuclei of granule cells, but none was seen in Purkinje cells. The alpha 2 fluorescence in testis was high, consistent with the high levels of alpha 2 mRNA in this tissue. The fluorescent signal appeared to originate primarily in dividing spermatogonia. Our findings support the concept that the beta 1 receptor plays a central role in T3-induced brain development and strongly suggest that the Purkinje cell is a direct target for T3.

Relationship of c-erbA mRNA content to tissue triiodothyronine nuclear binding capacity and function in developing and adult rats.

We have quantitated in adult and developing rat tissues the molar concentrations of c-erbA alpha 1- and beta 1-mRNAs, which code for nuclear T3-binding proteins, and c-erbA alpha 2-mRNA, which is generated by alternate splicing of the alpha gene transcript and codes for a receptor variant that does not bind T3. Comparison of the concentrations of c-erbA alpha 1-mRNA, beta 1-mRNA, or their sum to the T3 nuclear binding capacity per mg of DNA in adult liver, kidney, heart, cerebrum, and cerebellum and during the ontogeny of liver and brain shows that the T3 binding capacity/c-erbA mRNA ratio is tissue-specific and related to developmental state. Administration of T3 resulted in a 40-50% fall in the alpha 1 signal of adult liver, kidney, and heart without changing either the beta 1 signal or T3 binding capacity. A 40-fold increase in rat brain beta 1-mRNA occurred in the transition between the 19-day gestational fetus and the 10-day-old neonate. This corresponds to the period during which the T3 content rises in brain and during which T3 is known to influence central nervous system development. Our findings indicate that important translational or post-translational factors influence nuclear binding capacity and raise the possibility that c-erbA beta 1 may play a primary role in mediating T3 effects in developing and adult animals.

Binding of 3,5,3'-triiodothyronine (T3) and its analogs to the in vitro translational products of c-erbA protooncogenes: differences in the affinity of the alpha- and beta-forms for the acetic acid analog and failure of the human testis and kidney alpha-2 products to bind T3.

We have compared the affinities for T3 and the T3 analog binding characteristics of the in vitro translational products of seven c-erbA cDNAs (chicken c-erbA alpha; human placental c-erbA beta; rat c-erbA beta-1; rat c-erbA alpha-1; rat c-erbA alpha-2; human testis c-erbA alpha-2; and human kidney c-erbA alpha-2). Four of these (chicken c-erbA alpha, human placental c-erbA beta, rat c-erbA beta-1, rat c-erbA alpha-1) bound T3 with high affinity as previously described. When compared under identical conditions of synthesis and [125I]T3 binding, there was no significant difference between the affinity of the chicken c-erb A alpha-1 and the human c-erbA beta but in a more limited series the affinity of rat c-erbA beta-1 for T3 was 4.6-fold higher than that of the rat c-erbA alpha-1. In vitro translational products of the beta-probes showed a characteristic 2.2-fold higher triiodothyroacetic acid/T3 ratio than did the products of the alpha-probes, regardless of the species of origin of the probe. As previously established, the rat c-erbA alpha-2 product did not bind T3. However, in contrast to two published reports, the human testis and kidney alpha-2 probe products also failed to bind T3. These findings indicate that highly conserved C-terminal 37-40 residues are important for high affinity T3 binding by proteins encoded by the c-erb A family of genes.