We studied thyroid tissue from two siblings with Pendred's syndrome (familial goiter and congenital deafness), both with the Mondini-type inner ear malformation, goiter, and hypothyroidism. Iodine trapping and peroxidase levels were grossly normal. Thyroglobulin (Tg), the only iodoprotein found, had a normal monomer size (330 kilodaltons), but low content of hormone and iodine. Tg's expected N-terminal peptides of 26 and 18 kilodaltons, usually formed in association with iodination and thyroid hormone synthesis, were absent, but appeared after iodination in vitro. Reverse transcription of ribonucleic acid from Pendred thyroid tissue and amplification by polymerase chain reaction of specific regions encoding the most important hormonogenic sites of Tg revealed a normal complementary DNA sequence corresponding to the first 100 amino acid residues in Tg's N-terminus. However, 3 of 35 clones of the 3'-region corresponding to the Tg C-terminus exhibited a deletion of nucleotides 7860-7994; this deletion was not present in any of the 150 clones from 7 other thyroids we examined. Four Pendred clones had a 2-nucleotide deletion at positions 7870-7871, a change that would result in a premature stop codon and was found in thyroids from several other subjects as well. We conclude that the messenger ribonucleic acid encoding the 3'-region of Tg can be abnormal in Pendred's syndrome. Some, but not all, of these changes also occur in other human thyroids. Further work is necessary to show if and how these alterations relate to defective hormone synthesis and goiter.

Transplantation of GH3 rat pituitary tumor cells that express both PRL and GH to female Wistar-Furth rats results in tumors that secrete only GH. We have used in vivo passage of GH3 cells as a model system to study specific repression of PRL. RNA blot hybridization revealed that PRL message was repressed 95% in cells transplanted to host animals compared to that in GH3 cells in culture. In contrast, there was little change in GH message in the transplanted cells, and there was a 4-fold increase in insulin-like growth factor-I transcript levels. When the transplanted cells were returned to cell culture, PRL mRNA levels increased rapidly, reaching levels similar to those in GH3 cells within 72 h. Gene transfer studies demonstrated a low level PRL promoter utilization in GH3 cells after in vivo passage, when endogenous PRL was repressed. Transfection of the transplanted cells maintained in culture for 96 h, when endogenous PRL was expressed, demonstrated increased PRL promoter activity. Messenger RNA levels for the transcription factor Pit-1 were equivalent in GH3 cells and cells after in vivo passage, and the presence of Pit-1 protein in extracts from transplanted cells was demonstrated by Western blot analysis. Electrophoretic gel mobility shift assays indicated that protein interactions with the PRL promoter were very different for extracts prepared from cells in which PRL was repressed compared to those from cells maintained in culture until PRL expression had recovered.(ABSTRACT TRUNCATED AT 250 WORDS)

The pituitary-specific transcription factor Pit-1 is required for expression of the PRL gene. Transcription of the PRL gene in the anterior pituitary is both activated and repressed in response to neuroendocrine signals. The molecular events that mediate repression are unknown. Transplantation of GH3 pituitary tumor cells from culture to female Wistar-Furth rats resulted in repression of PRL gene expression. When the transplanted cells were returned to culture, PRL gene expression was rapidly activated. We used this model to study potential mechanisms by which PRL gene expression was silenced. In addition to the appropriate size Pit-1 proteins of 33 and 31 kilodaltons, smaller forms of the transcription factor, migrating at approximately 27 and 24 kilodaltons, were found in transplanted cells in which PRL gene expression was repressed. These smaller forms of Pit-1 protein were observed to disappear when transplanted cells were returned to culture, coincident with the activation of PRL gene expression. A transcript approximately 170 base pairs shorter than expected for that encoding full-length Pit-1 was detected in transplanted GH3 cell RNA by polymerase chain reaction. The shorter Pit-1 transcript was abundant only in GH3 cells after in vivo passage and was not readily detected in transplanted cells after as little as 12 h in culture. This shorter transcript was found to result from excision of sequence corresponding to exon IV and encodes a Pit-1 protein lacking 54 amino acids of the POU-specific domain. Gene transfer studies demonstrated this alternative form of Pit-1 inhibited PRL promoter activity.(ABSTRACT TRUNCATED AT 250 WORDS)

GH induces hepatic IGF-I synthesis by increasing transcription of its gene. IGF-I is synthesized, however, in many other tissues where the effect of GH on its gene expression is less well characterized. IGF-I and GH are produced by human lymphocytes and may function as autocrine regulators of lymphoproliferation. We have therefore used the human IM9 lymphocyte cell line to (A) define the IGF-I gene transcripts expressed and (B) investigate the effect of GH on early (protein tyrosine phosphorylation) and late (changes in IGF-I mRNA levels) events in intracellular signal transduction. Multiple IGF-I mRNA species, ranging in size from 0.9 to 5.8 kb, were detected by Northern hybridization of poly(A)+ mRNA from IM9 cells. The human IGF-I gene contains at least six exons and alternative splicing produces a number of transcripts. Solution hybridization with exon-specific riboprobes and amplification by PCR using exon-specific primers revealed that multiple transcripts were expressed in IM9 cells, and that exon 2 was the dominant leader exon. Treatment of IM9 cells with 200 ng recombinant human (rh)GH/ml led to the specific tyrosine phosphorylation of three intracellular proteins (93, 120 and 134 kDa), which are involved in the initial signalling of the GH transduction pathway. However a solution hybridization assay using the IGF-IA specific riboprobe on IM9 cell RNA from similar experiments revealed that GH treatment did not change IGF-I gene expression. This study has demonstrated (A) that the IGF-I gene is expressed in human IM9 lymphocytes, (B) that in contrast to other human tissue, exon 2 is the major leader exon, and (C) that rhGH induces tyrosine phosphorylation of 93, 120 and 134 kDa proteins but does not alter IGF-I gene expression. The IM9 cell may form an important model to investigate a GH transduction pathway not coupled to the IGF-I gene.

The present studies were designed to investigate the nature of the actions of insulin-like growth factor-II (IGF-II) on granulosa cell steroidogenesis and assess the potential facilitative interactions between IGF-II and other major regulators of ovarian sterol metabolism, viz. estrogen, FSH, and low density lipoprotein (LDL). In serum-free first passage monolayer cultures of swine granulosa cells, human recombinant IGF-II stimulated progesterone production with a half-maximally effective concentration of 4.6 +/- 1.2 ng/ml (0.61 +/- 0.16 nM) between 0-48 h of culture and 27 +/- 5.7 ng/ml (3.6 +/- 0.76 nM) between 48-96 h. Maximal progesterone accumulation increased 12-fold over that in untreated cultures (48-96 h). Over the latter interval, IGF-I stimulated progesterone production approximately 10-fold, with a significantly lower ED50 of 6.1 +/- 0.70 ng/ml (0.78 +/- 0.09 nM; P < 0.01 vs. IGF-II effect). IGF-II (100 ng/ml) enhanced progesterone biosynthesis approximately 2-fold in the presence of 25-hydroxycholesterol, suggesting that IGF-II increases the effective activity of the mitochondrial cholesterol side-chain cleavage enzyme. IGF-II (100 ng/ml) augmented human LDL-promoted progesterone production approximately 18-fold between 0-48 h of culture and approximately 6-fold between 48-96 h. In addition, IGF-II showed time-dependent stimulatory effects on the rates of [125I]iodo-LDL internalization, and the amounts of cell-associated and degraded lipoprotein. IGF-II increased by approximately 10-fold the number of specific high affinity LDL receptors on granulosa cells, with no apparent change in their binding affinity, as assessed in equilibrium competition studies. Coadministration of IGF-II and FSH (100 ng/ml) or estradiol (E2; 1 microgram/ml) for 2 days increased progesterone production synergistically. Cotreatment with FSH or E2 for 4 days decreased the ED50 of IGF-II's stimulation of progesterone accumulation by 61% and 50%, respectively (P < 0.01). Synergistic interactions also existed between IGF-II and 8-bromo-cAMP, which indicates that IGF-II can act in part at cellular loci distal to cAMP generation. Northern blot analysis of total RNA isolated from granulosa cells treated with IGF-II (100 ng/ml), FSH (100 ng/ml), or IGF-II plus FSH for 2 days revealed 5-, 7-, or 8-fold increases, respectively, in the amount of cytochrome P450 cholesterol side-chain cleavage enzyme mRNA. The same treatments produced 6-fold increases in the level of LDL receptor mRNA, as determined by solution hybridization/RNase protection assays.(ABSTRACT TRUNCATED AT 400 WORDS)

Pure heat-stable inhibitor of the cAMP-dependent protein kinase (PKI) has been isolated in high yield by using a bacterial expression vector constructed to synthesize the complete sequence of the rabbit muscle protein kinase inhibitor, plus an amino-terminal initiator methionine and glycine. Bacterially expressed PKI has an inhibitory activity identical to that of the protein isolated from rabbit skeletal muscle and, by gel filtration and gel electrophoresis, has the same physicochemical characteristics as the native physiological form of PKI. Fourier transformed infrared spectroscopy and CD establish that PKI has unusually large amounts of random coil and turn structures, with significantly smaller amounts of alpha-helix and beta structures.

The mechanisms responsible for decreased levels of cAMP-dependent protein kinase activity in a mutant Chinese hamster ovary cell line have been examined. The cAMP-resistant Chinese hamster ovary 10260 cell line was found to possess only 20% of the cAMP-dependent protein kinase activity found in wild-type cells. The presence of decreased concentrations of the catalytic subunit in these cells was confirmed through binding studies using a radiolabeled, heat-stable inhibitor of the kinase. Cloned Chinese hamster ovary catalytic subunit cDNAs were isolated, characterized, and used as hybridization probes to examine the relative concentrations of catalytic subunit mRNAs in the wild-type and 10260 cell lines. A 40-50% decrease in the concentration of the mRNA for the C alpha isozyme of the catalytic subunit was observed in 10260 cells, as compared with wild-type. This decrease in catalytic subunit mRNA concentration probably accounts for a portion of the decreased kinase activity in the mutant cells. Further analysis of C alpha mRNA by polymerase chain reaction confirmed the decreased expression of C alpha mRNA in 10260 cells and further demonstrated the presence of two different species of C alpha mRNA in the 10260 cells. One species of C alpha cDNAs was indistinguishable from the wild-type cDNA, but the other species was shorter. Nucleotide sequence analysis of the amplified cDNAs led to the identification of a 191-base pair deletion in the shorter cDNA. Gene transfer studies using wild-type and 10260 C alpha cDNAs demonstrated that the longer cDNA from the 10260 cells produced wild-type activity, but the shorter cDNA was inactive. These studies suggest that at least two alterations in gene expression are responsible for decreased cAMP-dependent protein kinase activity in the 10260 cell line. One alteration results in an approximately 2-fold decrease in the concentrations of C alpha mRNA in the cells. The other change produces two species of C alpha mRNA; one of the C alpha mRNAs does not encode an active kinase.

To begin analysis of the DNA sequences necessary for luteinizing hormone (LH) gene transcription, fusion genes containing the 5′ flanking region of the rat LHβ or the human α-subunit gene linked to luciferase were transfected into primary cultures of rat pituitary cells. The LHβ-luciferase construct was expressed in the primary cultures at a level 50 times greater than a promoterless luciferase control plasmid. Little or no expression of the LHβ-luciferase construct was detected following transfection of MCF-7, JAR or GH3 tumor cell lines. Treatment of transfected cells with gonadotropin-releasing hormone resulted in a modest induction of LHβ-luciferase activity. Considerably higher levels of LHβ-luciferase activity were obtained with cultures from ovariectomized rats than were obtained with cultures from intact female rats. Analysis of 5′ deletions of the LHβ-luciferase construct demonstrated that activity was well maintained even after substantial deletions. The shortest construct, which contained 75 base pairs of 5′ flanking sequence had 38% of the activity of the longest which contained 1.7 kilobase pairs of flanking sequence. These findings demonstrate that transfection of primary cultures of rat pituitary cells may provide a useful system for analysis of the cis-acting sequences and trans-acting factors required for LH gene expression.

To identify DNA regions important for basal and hormone-stimulated transcription of the rat PRL gene, a series of clustered point mutations were prepared within the immediate 5' flanking region. DNA fragments representing the wild-type and 19 different linker-scanner mutations of the PRL gene were each linked to a luciferase marker gene, and the DNA constructs were transferred into GH3 pituitary tumor cells by electroporation. Luciferase activity was determined 24 h after transfection in extracts from control cells or cells treated with 0.5 mM chlorophenylthio-cAMP, 100 nM TRH, or 100 nM phorbol myristate acetate. The individual clustered point mutations covered a region from just up-stream of the TATA box (position -30) to a position 193 basepairs up-stream from the start of transcription. Five regions in which mutations produced substantial decreases in both basal and cAMP-, TRH-, or phorbol ester-stimulated expression of the marker gene were detected. Three of these regions (positions -41 to -58, -113 to -124, and -149 to -156) correspond to previously identified binding sites for the pituitary-specific, homeobox protein, Pit-1/GHF-1. The fourth and fifth regions do not correspond to Pit-1/GHF-1-binding sites and presumably represent sites for an unidentified factor. Within these regions, sequences with some similarity to a consensus cAMP response element and an AP-2-binding site have been detected. These data confirm the importance of Pit-1/GHF-1 as a key factor in PRL gene transcription. In addition, the results suggest that additional transcription factors are probably required for efficient expression of the PRL gene.(ABSTRACT TRUNCATED AT 250 WORDS)