Different functions for the thyroid hormone receptors TRalpha and TRbeta in the control of thyroid hormone production and post-natal development

Par-4 is a widely expressed protein that sensitizes both prostatic and non-prostatic cells to apoptosis. Constitutive- or regulated- overexpression of Par-4 caused a reduction in the levels of the anti-apoptotic protein Bcl-2. Replenishment of Bcl-2 levels abrogated susceptibility to Par-4-dependent apoptosis, suggesting that Par-4-mediated apoptosis requires downmodulation of Bcl-2 levels. The inverse correlation between Par-4 and Bcl-2 expression was recapitulated in human prostate tumors. Par-4 but not Bcl-2 was detected in the secretory epithelium of benign prostatic tumors and in primary and metastatic prostate cancers that are apt to undergo apoptosis. Moreover, xenografts of human, androgen-dependent CWR22 tumors showed Par-4 but not Bcl-2 expression. By contrast, androgen-independent CWR22R tumors derived from the CWR22 xenografts showed mutually exclusive expression patterns of Par-4 and Bcl-2. These findings suggest a mechanism by which Par-4 may sensitize prostate tumor cells to apoptosis.     

Lack of coactivator interaction can be a mechanism for dominant negative activity by mutant thyroid hormone receptors

We studied the interactions of two natural thyroid hormone receptor (TR) mutants from patients with resistance to thyroid hormone (RTH) and an artificial TR mutant with a nuclear receptor corepressor, N-CoR, and a steroid receptor coactivator, SRC-1. In electrophoretic mobility shift assays, wild-type TRbeta-1 interacted with N-CoR in the absence of ligand, whereas T3 caused dissociation of the TRbeta-1/N-CoR complex and formation of TRbeta-1/SRC-1 complex. In contrast, a natural mutant (G345R) with poor T3-binding affinity formed TRbeta-1/N-CoR complex, both in the absence and presence of T3, but could not form TRbeta-1/SRC-1 complex. Another TR mutant, which bound T3 with normal affinity and containing a mutation in the AF-2 region (E457D), had normal interactions with N-CoR but could not bind SRC-1. Both these mutants had strong dominant negative activity on wild-type TR transactivation. Studies with a TR mutant that had slightly decreased T3-binding affinity (R320H) showed a T3-dependent decrease in binding to N-CoR and increase in binding to SRC-1 that reflected its decreased ligand binding affinity. Additionally, when N-CoR and SRC-1 were added to these receptors at various T3 concentrations in electrophoretic mobility shift assays, TR/N-CoR and TR/SRC-1 complexes, but not intermediate complexes were observed, suggesting that N-CoR release is necessary before SRC-1 binding to TR. Our data provide new insight on the molecular mechanisms of dominant negative activity in RTH and suggest that the inability of mutant TRs to interact with coactivators such as SRC-1, which results from reduced T3-binding affinity, is a determinant of dominant negative activity.     

Mutation in the thyroid hormone receptor beta gene (A317T) in a Thai subject with resistance to thyroid hormone

Analysis of the thyroid hormone receptor beta (TRbeta) gene of a Thai female with the syndrome of resistance to thyroid hormone (RTH) revealed a missense mutation at codon 317, changing the guanine in nucleotide 1234 to an adenine that results in the replacement of the normal alanine (GCT) with a threonine (ACT). The proposita was heterozygous, and this mutation was not present in her parents and her sister, compatible with a neomutation. This is the first report of TRbeta gene mutation causing RTH in an individual of Thai origin.     

Selective pituitary resistance to thyroid hormone produced by expression of a mutant thyroid hormone receptor beta gene in the pituitary gland of transgenic mice

Phosphine (PH3), from hydrolysis of metal phosphides, is an important insecticide (aluminum phosphide) and rodenticide (zinc phosphide) and is considered genotoxic and cytotoxic in mammals. This study tests the hypothesis that PH3-induced genotoxicity and cytotoxicity are associated with oxidative stress by examining liver (Hepa 1c1c7) cells for possible relationships among cell death, increases in reactive oxygen species (ROS) and lipid peroxidation, and elevated 8-hydroxyguanine (8-OH-Gua) in DNA. PH3 was generated from 0.5 mM magnesium phosphide (Mg3P2) to give 1 mM PH3 as the nominal and maximal concentration. This level causes 31% cell death at 6 h, measured by lactate dehydrogenase leakage, with appropriate dependence on concentration and time. The intracellular ROS level is elevated within 0.5 h following exposure to PH3, peaking at 235% of the control by about 1 h. Lipid peroxidation (measured as malondialdehyde plus 4-hydroxyalkenals) is increased up to 504% by PH3 at 6 h in a time-dependent manner. The level of 8-OH-Gua in DNA, a biomarker of mutagenic oxidative DNA damage analyzed by GC/MS, increases to 259% at 6 h after PH3 treatment. Antioxidants significantly attenuate the PH3-induced ROS formation, lipid peroxidation, 8-OH-Gua formation in DNA, and cell death, with the general order for effectiveness of GSH (5 mM) and D-mannitol (10 mM) (hydroxyl radical scavengers), then Tempol (2.5 mM) and sodium azide (3 mM) (superoxide anion and singlet oxygen scavengers, respectively). These studies support the hypothesis that PH3-induced mutagenic and cytotoxic effects are due to increased ROS levels, probably hydroxyl radicals, initiating oxidative damage.     

Identification of the second cluster of ligand-binding repeats in megalin as a site for receptor-ligand interactions

Megalin is a large cell surface receptor that mediates the binding and internalization of a number of structurally and functionally distinct ligands from the lipoprotein and protease:protease inhibitor families. To begin to address how megalin is able to bind ligands with unique structurally properties, we have mapped a binding site for apolipoprotein E (apoE)-beta very low density lipoprotein (beta VLDL), lipoprotein lipase, aprotinin, lactoferrin, and the receptor-associated protein (RAP) within the primary sequence of the receptor. RAP is known to inhibit the binding of all ligands to megalin. We identified a ligand-binding site on megalin by raising mAb against purified megalin, selected for a mAb whose binding to megalin is inhibited by RAP, and mapped the epitope for this mAb. mAb AC10 inhibited the binding of apoE-beta VLDL, lipoprotein lipase, aprotinin, and lactoferrin to megalin in a concentration-dependent manner. When cDNA fragments encoding the four cysteine-rich ligand-binding repeats in megalin were expressed in a baculovirus system and immunoblotted with AC10, it recognized only the second cluster of ligand-binding repeats. The location of the epitope recognized by mAb AC10 within this domain was pinpointed to amino acids 1111-1210. From these studies we conclude that the binding of apoE-beta VLDL, lactoferrin, aprotinin, lipoprotein lipase, and RAP to megalin is either competitively or sterically inhibited by mAb AC10 suggesting that these ligands bind to the same or closely overlapping sites within the second cluster of ligand-binding repeats.     

Activating mutations in the extracellular domain of the fibroblast growth factor receptor 2 function by disruption of the disulfide bond in the third immunoglobulin-like domain

Multiple human skeletal and craniosynostosis disorders, including Crouzon, Pfeiffer, Jackson-Weiss, and Apert syndromes, result from numerous point mutations in the extracellular region of fibroblast growth factor receptor 2 (FGFR2). Many of these mutations create a free cysteine residue that potentially leads to abnormal disulfide bond formation and receptor activation; however, for noncysteine mutations, the mechanism of receptor activation remains unclear. We examined the effect of two of these mutations, W290G and T341P, on receptor dimerization and activation. These mutations resulted in cellular transformation when expressed as FGFR2/Neu chimeric receptors. Additionally, in full-length FGFR2, the mutations induced receptor dimerization and elevated levels of tyrosine kinase activity. Interestingly, transformation by the chimeric receptors, dimerization, and enhanced kinase activity were all abolished if either the W290G or the T341P mutation was expressed in conjunction with mutations that eliminate the disulfide bond in the third immunoglobulin-like domain (Ig-3). These results demonstrate a requirement for the Ig-3 cysteine residues in the activation of FGFR2 by noncysteine mutations. Molecular modeling also reveals that noncysteine mutations may activate FGFR2 by altering the conformation of the Ig-3 domain near the disulfide bond, preventing the formation of an intramolecular bond. This allows the unbonded cysteine residues to participate in intermolecular disulfide bonding, resulting in constitutive activation of the receptor.     

Certain activating mutations within helix 6 of the human luteinizing hormone receptor may be explained by alterations that allow transmembrane regions to activate Gs

Male-limited gonadotropin-independent precocious puberty (MPP) is frequently associated with mutations of the human LH/CG receptor (hLHR) that result in constitutively active hLHRs. Many such activating mutations have been identified in transmembrane 6 of the hLHR, with the substitution of Asp-578 being the most frequently observed mutation. Mutagenesis of a transmembrane helix of a G protein-coupled receptor can cause local alterations in the conformation near the mutated residue, allosteric changes elsewhere in the protein, and/or changes in the interhelical packing of the receptor. Therefore, while it has been hypothesized that activation of the receptor by mutations of Asp-578 may arise via alterations in the interactions of helix 6 with other transmembrane helices and/or by allosterically altering the conformation of the third intracellular loop, it has not been possible to ascertain the role of the sixth transmembrane helix per se in activating Gs in the mutated full-length receptor. Recently, however, we have shown that a peptide KMAILIFT, corresponding to the juxtacytoplasmic portion of helix 6 of the hLHR, is capable of activating Gs. These results suggest that helix 6 itself can directly interact with Gs. Importantly, the KMAILIFT peptide did not include Asp-578, which lies just C-terminal to this sequence. We show herein that a peptide extended to include Asp-578 (KMAILIFTDFT) is a poor activator of Gs. However, if the peptide is synthesized with the aspartate replaced with either a glycine or tyrosine, substitutions that are found in some patients with MPP, these peptides have Gs-stimulating activity. Additionally, a transmembrane 6 peptide with the substitution of Ile-575 with leucine, another mutation found in MPP, mimicked the activating effects of this mutation in the full-length receptor. The ability of peptides in which Asp-578 or Ile-575 is substituted to mimic the activating effects of these mutations in the full-length receptor suggests that the sixth transmembrane helix represents a site for direct interaction with Gs. In addition to the stimulatory effects of transmembrane 6 peptides, peptides corresponding to the juxtacytoplasmic portions of the fourth, fifth, and seventh helices were also able to stimulate Gs. These results are consistent with the hypothesis that the transmembrane helices may form a pocket for interaction with Gs and that constitutive activation of the hLHR may involve the opening of the pocket formed by these helices, thus exposing Gs-binding sites on these helices.     

A monoclonal antibody to the ligand-binding domain of the neurokinin 1 receptor (NK1-R) for the neuropeptide substance P

For PCR-based genotyping using polymorphic microsatellite markers, DNA from decomposed postmortem human tissues was fractionated into six groups according to molecular size. The minimum required amounts of this degraded DNA, for detecting alleles at five microsatellite loci (ACTBP2, CMAG, HUMTH01, CYP19, and LPL) and one minisatellite locus (MCT118) were investigated respectively. The allele patterns were detected by electrophoresis of the PCR products on a 6%-denaturing polyacrylamide gel following silver staining. The detection of alleles for the loci with large allele size required more template DNA with higher molecular size than for that with small allele size. Amounts from 0.3 ng to 5 ng were needed for allele detection on genomic DNA from fresh blood. When the decomposed DNA mixture was used as the template, approximately ten times the amount of genomic DNA was required to detect alleles at the three loci of LPL, CYP19 and HUMTH01, while 24 to 67 times was required for the loci, CMAG, ACTBP2 and MCT118. It was demonstrated that a minimum molecular, size and amount of template DNA was needed for amplifying alleles of the six loci, and degraded DNA less than minimum size in the samples would prevent the detection of the loci which have large allele size.     

Skeletal resistance to the calcemic action of parathyroid hormone in uremia: role of 1,25 (OH)2 D3

Studies were carried out to investigate the role of 1,25(OH)2D3 in the skeletal resistance to the calcemic action of parathyroid hormone. The change in serum calcium after the intravenous infusion of 2 U of parathyroid extract (PTE)/kg body wt/hr for eight hours was evaluated in thyroparathyroidectomized (T-PTX) dogs before, and one, two and three days after, induction of uremia by bilateral ureteral ligation (11 dogs) or by bilateral nephrectomy (8 dogs). In another six nephrectomized and T-PTX dogs, 0.68 ug of 1, 25 (OH)2D3/day was given on the day of nephrectomy and for two days thereafter. Serum creatinine in each day of the study was not different among the three groups. The study also included the evaluation of the effect of sham operation (five dogs) and the administration of 1,25 (OH)2D3 to dogs with normal renal function (four dogs) on the calcemic response to PTE, as well as the reproducibility of such a response in the same animal. The results showed that 1) the calcemic response to PTE was markedly impaired after one day of bilateral ureteral ligation or nephrectomy, but the impairment was more severe after nephrectomy; 2) the calcemic response to PTE after two or three days of bilateral ureteral ligation was similar to that seen at one day after nephrectomy; 3) 1, 25 (OH)2D3 partially restored the calcemic response to PTE in the nephrectomized animals to levels similar to those seen after one day of bilateral ureteral ligation; 4) sham operation did not affect the response to PTE, and repeated infusion of PTE produced similar changes in the concentrations of serum calcium. The data indicate that (a) a deficiency of 1,25 (OH)2D3 is at least partly responsible for the skeletal resistance to the calcemic action of PTH in uremia; (b) uremia, per se, may also contribute to this phenomenon; and (c) the kidney after one day of complete bilateral ureteral ligation may still produce 1,25 (OH)2D3, but this ability is compromised after two days of ureteral obstruction.     

Missense mutations define a restricted segment in the C-terminal domain of phytochrome A critical to its regulatory activity

The phytochrome family of photoreceptors has dual molecular functions: photosensory, involving light signal perception, and regulatory, involving signal transfer to downstream transduction components. To define residues necessary specifically for the regulatory activity of phytochrome A (phyA), we undertook a genetic screen to identify Arabidopsis mutants producing wild-type levels of biologically defective but photochemically active and dimeric phyA molecules. Of eight such mutants identified, six contain missense mutations (including three in the same residue, glycine 727) clustered within a restricted segment in the C-terminal domain of the polypeptide. Quantitative photobiological analysis revealed retention of varying degrees of partial activity among the different alleles--a result consistent with the extent of conservation at the position mutated. Together with additional data, these results indicate that the photoreceptor subdomain identified here is critical to the regulatory activity of both phyA and phyB.     

Helix 2 of the paired domain plays a key role in the regulation of DNA-binding by the Pax-3 homeodomain

Pax3 contains two structurally independent DNA-binding domains, a paired domain (PD) and a homeodomain (HD). Biochemical and mutagenesis studies have shown that both domains are functionally interdependent. In particular, it has been shown that the PD can regulate the DNA-binding specificity and dimerization potential of the HD. To delineate Pax3 protein segments that are involved in the regulation of HD DNA-binding, a series of chimeric proteins were created in which the HD and linker region were gradually replaced with corresponding sequences from a heterologous HD protein, Phox. Characterization of chimeric proteins by electrophoretic mobility shift analysis (EMSA) suggests that a portion of the linker region contributes to the functional interaction between the PD and HD. In addition, stepwise removal of sequences from the Pax3 PD was used to define regions within this domain that are involved in the regulation of HD DNA-binding. EMSA of these proteins in the context of the chimeric Pax3/Phox backbone provided two key findings: (i) the C-terminal subdomain of the PD does not play a major role in the regulation of HD DNA-binding and (ii) the N-terminal subdomain and, in particular, the second alpha-helix are essential for modulation of HD DNA-binding. Significantly, deletion of helix 2 was found to be sufficient to uncouple regulation of HD DNA-binding by the PD.     

A minimal role for selectins in the recruitment of leukocytes into the inflamed liver microvasculature

A two-step paradigm for leukocyte recruitment has been established in a number of tissues including the mesentery, skin, and muscle, and necessitates an initial rolling step via the selectins before firm leukocyte adhesion via the integrins. In view of the many inflammatory diseases that involve the liver, we investigated the importance of rolling and the selectins in the hepatic microvasculature and compared the responses to that of the commonly used mesentery or cremaster microvasculature. We visualized the liver microvasculature using intravital microscopy and we determined that within the liver the majority of leukocytes adhere within the sinusoids (80%) in response to a chemotactic stimulus such as FMLP (20% in postsinusoidal venules) whereas leukocytes adhere exclusively within postcapillary venules in tissue like the mouse cremaster. In the sinusoids, the adhesive response to FMLP is not dependent upon selectins inasmuch as adhesion was not reduced in the sinusoidal vessels of P-selectin-deficient mice or E-selectin/P-selectin- deficient animals in the presence or absence of L-selectin antibody. No rolling or adhesion was detected in response to FMLP in the selectin-deficient cremaster microvasculature. Immunoneutralization of selectins with fucoidan in wildtype mice eliminated rolling and adhesion in the cremaster but failed to affect adhesion in the liver sinusoids in response to FMLP. More long-term leukocyte recruitment with lipopolysaccharide (4 h) was also impaired in the cremaster but not the liver microvasculature in selectin-deficient animals. Leukocyte adhesion in the sinusoids was reduced in P-selectin-deficient mice also lacking intercellular adhesion molecule-1 (ICAM-1). This study for the first time demonstrates that selectins are not an essential step for leukocyte recruitment into the inflamed liver microvasculature.     

A role for thyroid hormones in the regulation of diet-induced thermogenesis in birds

The possible involvement of thyroid hormones in avian diet-induced thermogenesis (DIT) was investigated in two lines of cockerels divergently selected for high (R-) or low (R+) food efficiency. For a given body weight, R+ cockerels exhibited a higher food intake than R- cockerels (+49 to +76%) and increased DIT (+25%). Plasma thyroxine (T4) level did not differ between lines whatever the feeding status of the birds. Plasma 3,5,3'-triiodothyronine (T3) level was lower in fasted R+ than in fasted R- cockerels while the opposite was observed after a meal. Iopanic acid injections reduced both plasma T3 concentrations and heat production to the same levels in both lines. Hepatic 5'-deiodinase activity measured with an exogenous sulfhydryl group (dithiothreitol) did not differ between lines, but when the sulfhydryl group was omitted, the activity was higher in R+ than in R- birds (90 v. 42 pmol T3/min per liver). T3-binding capacity of isolated hepatic nuclei was higher (+76%) in R+ than in R- birds. Long-term or acute pair-feeding of R+ cockerels to the level of R- controls did not alter these results. The present results suggest that T3, mainly originating from peripheral conversion of T4 to T3, is involved in DIT in the R+ line. Availability of endogenous sulfhydryl groups appears to play an important part in the modulation of hepatic deiodinase activity. The higher concentration of nuclear T3 receptors may further increase the effects of the hormone, suggesting a major role of thyroid hormones associated with catecholamines in the stimulation of avian DIT. The underlying thermogenic mechanisms remain to be elucidated.