Thyroxine promotes association of mitogen-activated protein kinase and nuclear thyroid hormone receptor (TR) and causes serine phosphorylation of TR.

Activated nongenomically by l-thyroxine (T(4)), mitogen-activated protein kinase (MAPK) complexed in 10-20 min with endogenous nuclear thyroid hormone receptor (TRbeta1 or TR) in nuclear fractions of 293T cells, resulting in serine phosphorylation of TR. Treatment of cells with the MAPK kinase inhibitor, PD 98059, prevented both T(4)-induced nuclear MAPK-TR co-immunoprecipitation and serine phosphorylation of TR. T(4) treatment caused dissociation of TR and SMRT (silencing mediator of retinoid and thyroid hormone receptor), an effect also inhibited by PD 98059 and presumptively a result of association of nuclear MAPK with TR. Transfection into CV-1 cells of TR gene constructs in which one or both zinc fingers in the TR DNA-binding domain were replaced with those from the glucocorticoid receptor localized the site of TR phosphorylation by T(4)-activated MAPK to a serine in the second zinc finger of the TR DNA-binding domain. In an in vitro cell- and hormone-free system, purified activated MAPK phosphorylated recombinant human TRbeta1 (). Thus, T(4) activates MAPK and causes MAPK-mediated serine phosphorylation of TRbeta1 and dissociation of TR and the co-repressor SMRT.

Thyroid hormone induces activation of mitogen-activated protein kinase in cultured cells.

Thyroid hormone [L-thyroxine (T4)] rapidly induced phosphorylation and nuclear translocation (activation) of mitogen-activated protein kinase (MAPK) in HeLa and CV-1 cells in the absence of cytokine or growth factor. A pertussis toxin-sensitive and guanosine 5'-O-(3-thiotriphosphate)-sensitive cell surface mechanism responsive to T4 and agarose-T4, suggesting a G protein-coupled receptor, was implicated. Cells depleted of MAPK or treated with MAPK pathway inhibitors showed reduced activation of MAPK and of the signal transducer and activator of transcription STAT1alpha by T4; they also showed reduced T4 potentiation of the antiviral action of interferon-gamma (IFN-gamma). T4 treatment caused tyrosine-phosphorylated MAPK-STAT1alpha nuclear complex formation and enhanced Ser-727 phosphorylation of STAT1alpha, in the presence or absence of IFN-gamma. STAT1alpha-deficient cells transfected with STAT1alpha containing an alanine-for-serine substitution at residue 727 (STAT1alphaA727) showed minimal T4-stimulated STAT1alpha activation. IFN-gamma induced the antiviral state in cells containing wild-type STAT1alpha (STAT1alphawt) or STAT1alphaA727; T4 potentiated IFN-gamma action in STAT1alphawt cells but not in STAT1alphaA727 cells. T4-directed STAT1alpha Ser-727 phosphorylation is MAPK mediated and results in potentiated STAT1alpha activation and enhanced IFN-gamma activity.

Potentiation by thyroid hormone of human IFN-gamma-induced HLA-DR expression.

We have investigated the mechanism by which thyroid hormone potentiates IFN-gamma-induced HLA-DR expression. IFN-gamma-induced HLA-DR expression requires activation of STAT1alpha and induction of the Class II trans-activator, CIITA. HeLa and CV-1 cells treated only with L-thyroxine (T4) demonstrated increased tyrosine phosphorylation and nuclear translocation (= activation) of STAT1alpha; this hormone effect on signal transduction, and T4 potentiation of IFN-gamma-induced HLA-DR expression, were blocked by the inhibitors CGP 41251 (PKC) and genistein (tyrosine kinase). Treatment of cells with T4-agarose also caused activation of STAT1alpha. In the presence of IFN-gamma, T4 enhanced cytokine-induced STAT1alpha activation. Potentiation by T4 of IFN-gamma action was associated with increased mRNA for both CIITA and HLA-DR, with peak enhancement at 16 h (CIITA), and 2 d (HLA-DR). T4 increased IFN-gamma-induced HLA-DR protein 2.2-fold and HLA-DR mRNA fourfold after 2 d. Treatment with actinomycin D after induction of HLA-DR mRNA with IFN-gamma, with or without T4, showed that thyroid hormone decreased the t(1/2) of mRNA from 2.4 to 1.1 h. HeLa and CV-1 cells lack functional nuclear thyroid hormone receptor. Tetraiodothyroacetic acid (tetrac) and 3,5,3'-triiodo-thyroacetic acid (triac) blocked T4 potentiation of IFN-gamma-induced HLA-DR expression and T4 activation of STAT1alpha. These studies define an early hormone recognition step at the cell surface that is novel, distinct from nuclear thyroid hormone receptor, and blocked by tetrac and triac. Thus, thyroid hormone potentiation of IFN-gamma-induced HLA-DR transcription is mediated by a cell membrane hormone binding site, enhanced activation of STAT1alpha, and increased CIITA induction.

Posterior approaches to the humerus: when should you worry about the radial nerve?

We used the olecranon osteotomy approach to the humerus on 75 cadaver arms and measured where the radial nerve pierced the intermuscular septum to determine the risk to that nerve during elevation of the triceps. We found the nerve an average of 10.0 cm from the distal articular surface in men and 9.4 cm in women; however, some cadavers had nerves as close as 7.5 cm. The humerus was also approached via a triceps split and the nerve located at the spiral groove. The distance from the articular surface to the nerve averaged 15.8 cm in men and 15.2 cm in women, with the minimum distance being 13 cm in one woman. When dissection beyond 7.5 cm laterally or 13.0 cm posteriorly is required, care should be taken to isolate and protect the radial nerve.

Potentiation by thyroxine of interferon-gamma-induced HLA-DR expression is protein kinase A- and C-dependent.

L-Thyroxine (T4) and 3,3',5-L-triiodothyronine (T3) potentiate the antiviral state induced by interferon-gamma(IFN-gamma) in homologous cells by a mechanism that is dependent upon calcium/phospholipid-dependent protein kinase (PKC). L-T4 and T3 also potentiate induction by IFN-gamma of MHC class II HLA-DR antigen expression in HeLa cells. In the present studies of HLA-DR expression, the PKC inhibitor staurosporine (0.1-1 nM) enhanced the expression of HLA-DR when the inhibitor was added simultaneously with IFN-gamma, 100 IU/ml. In the presence of IFN-gamma and 10(-7) M T4, the same concentrations of staurosporine inhibited potentiation of HLA-DR expression by thyroid hormone. A more specific PKC inhibitor, CGP41251 (0.5-5 nM), similarly enhanced HLA-DR expression in the presence of IFN-gamma but inhibited thyroid hormone potentiation of antigen expression. Both actions of CGP41251 were suppressed when cells were also treated with phorbol 12-myristate 13-acetate (PMA). A phospholipase C inhibitor, U73122 (1-1000 nM), did not alter the potentiating ability of T4, although it inhibited in a concentration-dependent manner the expression of HLA-DR induced by IFN-gamma. The potentiating effect of T4 was much more sensitive to a cyclic AMP-dependent protein kinase (PKA) inhibitor,KT5720 (1-1000nM), than was the induction of HLA-DR by IFN-gamma. The inhibitory effects of KT5720 were reversed by concurrent 8-bromo-cAMP treatment. The calmodulin antagonist W-7 (5-50 microM) did not alter IFN-gamma induction of HLA-DR in either the presence or absence of T4. HLA-DR expression in HeLa cells appears to be under PKC-associated inhibition; IFN-gamma reverses this inhibition to promote the appearance of the DR antigen. In contrast, potentiation by T4 of induction of HLA-DR by IFN-gamma requires activation of PKC. PKA is involved both in DR induction by IFN-gamma and in potentiation of the latter by T4. Thus, PKA and PKC have discrete roles in IFN-gamma-induced MHC class II antigen expression and its modulation by thyroid hormone.

Transforming growth factor-beta induces plasminogen activator inhibitor type-1 in cultured human orbital fibroblasts.

PURPOSE: The level of constitutive plasminogen activator inhibitor type-1 (PAI-1) expression in cultured human orbital fibroblasts is considerably lower than that found in dermal fibroblasts. This divergence in PAI-1 expression implies differences in the pericellular proteolytic environment and, therefore, in the turnover of extracellular matrix. In this article, the authors examine the effect of transforming growth factor-beta (TGF-beta) on PAI-1 expression in orbital fibroblasts. METHODS: Human orbital and dermal fibroblasts were grown in culture. Confluent monolayers were treated with TGF-beta. PAI-1 in the extracellular matrix was quantitated by radiolabeling the cultures and electrophoresing the cellular material on SDS-PAGE. Medium content was determined by immunoprecipitation of [35S]PAI-1 with a rabbit, anti-human, polyclonal antibody. PAI-1 mRNA was determined by Northern hybridization. RESULTS: TGF-beta increased PAI-1 levels in orbital fibroblasts in a dose-dependent manner, up to 35-fold. The induction was maximal after 16 hours of treatment. The increases in extracellular matrix PAI-1 paralleled those observed in the medium. The steady state levels of the mRNA encoding the protein were upregulated by TGF-beta up to 60-fold 8 hours after the addition of TGF-beta. The fractional increase in PAI-1 expression in orbital fibroblasts was consistently greater than that observed in dermal strains. CONCLUSIONS: Exposure to TGF-beta consistently induces PAI-1 expression in orbital fibroblasts, cells that do not express the polypeptide constitutively at high levels. The effects are mediated at the pretranslational level and involve the upregulation of PAI-1 mRNA. These results suggest that TGF-beta may exert a profound regulatory influence on the pericellular proteolytic environment in orbital connective tissue.

Viability of intramedullary canal bone reamings for continued calcification.

The viability of intramedullary canal bone reamings as a potential bone graft material was examined. Intramedullary bone reamings were obtained from the tibia or femur of three patients during intramedullary nailing procedures. Histologic examination showed bone marrow elements with complete disruption of the marrow compartmental organization. Bone trabeculas were present in a randomly scattered fashion with no structural organization. To assess the viability after reaming of the material for continued calcification, implants of the reaming material were placed in a pocket made in the gluteus maximus muscle of 12 rats. After 7 days, the implanted bone spicules showed evidence of tetracycline label uptake, indicating the material obtained after reaming the intramedullary canal was still viable and capable of continued calcification. That the intramedullary canal bone reamings may be a source of bone graft material is interesting. The reamings appear to continue an osteoproductive capacity when used as a graft.

The presence of intranuclear lipid inclusions in hepatocytes of mice after chronic ingestion of polybrominated biphenyl.

Electron microscopic study of livers from mice fed 167 ppm polybrominated biphenyl (PBB) for 12 weeks showed hepatocytes with nuclei containing varied amounts of lipid inclusions. The inclusions appeared as spherical vacuoles free in the nuclear matrix. This is the first report of the induction of lipid inclusions within the nucleus by a halogenated hydrocarbon.

The presence of hepatic intramitochondrial crystalline inclusions in polybrominated biphenyl-treated mice.

Electron microscopic study of livers from mice fed 167 ppm polybrominated biphenyl (PBB) revealed mitochondrial abnormalities which consisted of both alterations in size and the formation of crystalline-like inclusions within the mitochondrial matrix. These inclusions appeared as parallel arrays of rods and were found in elongated mitochondria which contained few cristae. The findings are briefly described and the possible significance of such inclusions in relation to mitochondrial aberrations are discussed.

Unconditional-stimulus locus and interstimulus-interval shift in rabbit (Oryctolagus cuniculus) nictitating membrane conditioning.

The nictitating membrane response of rabbits was conditioned at a 200 msec interstimulus interval (ISI) with either circumorbital (C) or paraorbital (P) shock as the unconditional-stimulus locus. After 3 acquisition days half of each group was shifted to a 700 msec interstimuls interval. Results indicated: (1) more rapid acquisition for Group C, (2) postshift response decrements for both groups, (3) more rapid and stable, as well as complete return to preshift performance levels for Group C. Results were discussed in terms of the response-shaping hypothesis and the contiguity-substitution hypothesis in explaining both conditional response emergence and subsequent modifications of CR topography.

Growth of a plasma cell myeloma in lathyritic mice.

Growth of a plasma cell myeloma (Adj PC-5) was studied in mice made lathyritic by the administration of beta-amino-proprionitrile (BAPN). The number of bones that had their medually cavities filled with tumor cells was notably decreased compared with tumor-bearing mice not treated with BAPN. Other aspects of tumor growth were the same. BAPN caused some retardation of tumor growth in the medullary cavity, but also caused osteoporosis and decreased tensile strength of collagen that allowed expansion of tumor growth outside the bones to proceed as usual. Additionally, the tumor-bearing mice showed some inhibition in their response to BAPN. This agrees with previous work that has shown that the response to BAPN treatment is greatest in animals that otherwise are healthiest.