Antagonistic effects of thyrotropin and epidermal growth factor on thyroglobulin mRNA level in cultured thyroid cells

Both thyrotropin (TSH) and epidermal growth factor (EGF) are potent mitogenic agents when added to dog thyroid cells in primary culture [Roger, P. P. and Dumont, J. E. (1984) Mol. Cell. Endocrinol. 36, 79-93]. The concomitant effect of these agents on the differentiation state of the cells was appreciated using cell morphology, iodide trapping, thyroglobulin synthesis and cytoplasmic thyroglobulin mRNA content as markers. Together with previous results [Mol. Cell. Endocrinol. 36, 79-93 (1984)] it is shown that cells cultured in the continuous presence of TSH maintain all the parameters at a near normal level. In the absence of TSH, thyroglobulin mRNA decreased to very low, though still detectable levels. Addition of TSH restored subnormal mRNA levels. Culture of cells in the presence of EGF for 4-6 days affected profoundly their morphology, abolished iodide trapping and decreased thyroglobulin synthesis and cytoplasmic mRNA content to undetectable levels. Addition of TSH to cells previously exposed to EGF reversed the growth factor effect on all four indexes. The redifferentiating effect of TSH was well observed within 3-4 days and was mimicked by the adenylate cyclase activators, forskolin and cholera toxin. When administered simultaneously, TSH and EGF achieved an intermediate situation, EGF antagonizing partially the effect of TSH on the expression of thyroglobulin gene. Another growth factor, fibroblast growth factor, while promoting thyroid cell proliferation also, did not interfere at all with TSH effects on cytoplasmic thyroglobulin mRNA content. Our results make the dog thyroid cell in primary culture an appropriate model to study the mechanisms involved in gene regulation by cyclic AMP and growth factors.     

Involvement of 70-kD heat-shock proteins in peroxisomal import

This report describes the involvement of 70-kD heat-shock proteins (hsp70) in the import of proteins into mammalian peroxisomes. Employing a microinjection-based assay (Walton, P. A., S. J. Gould, J. R. Feramisco, and S. Subramani. 1992. Mol. Cell Biol. 12:531-541), we demonstrate that proteins of the hsp70 family were associated with proteins being imported into the peroxisomal matrix. Import of peroxisomal proteins could be inhibited by coinjection of antibodies directed against the constitutive hsp70 proteins (hsp73). In a permeabilized-cell assay (Wendland and Subramani. 1993. J. Cell Biol. 120:675-685), antibodies directed against hsp70 proteins were shown to inhibit peroxisomal protein import. Inhibition could be overcome by the addition of exogenous hsp70 proteins. Purified rat liver peroxisomes were shown to have associated hsp70 proteins. The amount of associated hsp70 was increased under conditions of peroxisomal proliferation. Furthermore, proteinase protection assays indicated that the hsp70 molecules were located on the outside of the peroxisomal membrane. Finally, the process of heat-shocking cells resulted in a considerable delay in the import of peroxisomal proteins. Taken together, these results indicate that heat-shock proteins of the cytoplasmic hsp70 family are involved in the import of peroxisomal proteins.     

The Xenopus laevis aurora-related protein kinase pEg2 associates with and phosphorylates the kinesin-related protein XlEg5.

We have previously reported on the cloning of XlEg5, a Xenopus laevis kinesin-related protein from the bimC family (Le Guellec, R., Paris, J., Couturier, A., Roghi, C., and Philippe, M. (1991) Mol. Cell. Biol. 11, 3395-3408) as well as pEg2, an Aurora-related serine/threonine kinase (Roghi, C., Giet, R., Uzbekov, R., Morin, N., Chartrain, I., Le Guellec, R., Couturier, A., Dorée, M., Philippe, M., and Prigent, C. (1998) J. Cell Sci. 111, 557-572). Inhibition of either XlEg5 or pEg2 activity during mitosis in Xenopus egg extract led to monopolar spindle formation. Here, we report that in Xenopus XL2 cells, pEg2 and XlEg5 are both confined to separated centrosomes in prophase, and then to the microtubule spindle poles. We also show that pEg2 co-immunoprecipitates with XlEg5 from egg extracts and XL2 cell lysates. Both proteins can directly interact in vitro, but also through the two-hybrid system. Furthermore immunoprecipitated pEg2 were found to remain active when bound to the beads and phosphorylate XlEg5 present in the precipitate. Two-dimensional mapping of XlEg5 tryptic peptides phosphorylated in vivo first confirmed that XlEg5 was phosphorylated by p34(cdc2) and next revealed that in vitro pEg2 kinase phosphorylated XlEg5 on the same stalk domain serine residue that was phosphorylated in metabolically labeled XL2 cells. The kinesin-related XlEg5 is to our knowledge the first in vivo substrate ever reported for an Aurora-related kinase.     

Synthetic substrate for eukaryotic signal peptidase. Cleavage of a synthetic peptide analog of the precursor region of preproparathyroid hormone

A synthetic peptide analog of the precursor region of preproparathyroid hormone has been shown to be a specific substrate for hen oviduct signal peptidase. The sequence of the 31-residue peptide is Ser-Ala-Lys-Asp-norleucine (Nle)-Val-Lys-Val-Nle-Ile-Val-Nle-Leu-Ala-Ile-Ala-Phe-Leu-Ala-Arg-Ser-As p-Gly-Lys-Ser-Val-Lys-Lys-Arg-D-Tyr-amide (Caulfield, M. P., Duong, L. T., O'Brien, R., Majzoub, J. A., and Rosenblatt, M. (1988) Mol. Endocrinol. 2, 452-458). This sulfur-free signal peptide analog can be labeled with 125I on the C-terminal D-tyrosine and is cleaved by purified hen oviduct signal peptidase between Gly and Lys, the correct site of cleavage of preproparathyroid hormone in vivo. Amino acid sequence analysis of the cleavage product released 125I at the seventh cycle of Edman degradation, confirming that enzymatic cleavage occurs at the physiological site. Synthetic peptide analogs of the substrate with Lys, Pro, or Asp substituted for Nle-18 were poor substrates for the enzyme and were also poor competitive inhibitors of catalysis, suggesting that modifications at position -18, 12 amino acids from the site of cleavage, directly influence binding by the enzyme. Analysis of the reactivity of signal peptidase with these synthetic peptides provides insight into the cleavage specificity requirements of this eukaryotic signal peptidase.     

Do calcium and calmodulin trigger maturation in amphibian oocytes?

In contrast to earlier reports (J. L. Maller and E. G. Krebs, 1980, Curr. Top. Cell. Regul. 16, 271-311; M. Moreau, J. P. Vilian, and P. Guerrier, 1980, Dev. Biol. 78, 201-214; W. J. Wasserman and L. D. Smith, 1981, J. Cell Biol. 89, 389-394; D. Huchon, R. Ozon, E. H. Fischer, and J. G. Demaille, 1981, Mol. Cell. Endocrinol. 22, 211-222) calmodulin preparations isolated from Xenopus laevis ovaries or obtained commercially rarely induced maturation upon microinjection into individual oocytes. Calmodulin injections did result in significant cases of maturation when oocytes were first pretreated (primed) with calcium-free (EGTA) OR-2 and then injected in regular OR-2 medium. However, under these conditions the injected buffer solution alone was sometimes found to induce maturation. Under more optimal priming conditions, cases were found where as high as 100% of the oocytes matured simply by returning them to regular OR-2 medium. To determine which divalent cations could be involved in the priming effects of EGTA pretreatment we repeated the earlier ionophore work of W. J. Wasserman and Y. Masui (1975, J. Exp. Zool. 193, 369-375), looking not just at calcium and magnesium but other divalent cations as well. Several divalent cations (10 mM) were found to induce germinal vesicle breakdown with the following tentative order of efficacy, Co2+ greater than or equal to Zn2+ greater than or equal to Mn2+ greater than Ca2+ greater than Mg2+ greater than Ba2+, regardless of whether or not ionophore A23187 was present. These results, along with other reports in the literature, are discussed with respect to the theory that a rise in free calcium and calmodulin is involved in triggering oocyte maturation; we conclude that neither is involved.     

Xenopus egg lysates repair heat-generated DNA nicks with an average patch size of 36 nucleotides.

Base excision repair (BER) is an essential DNA repair pathway since it processes spontaneous (endogenous) DNA damage such as abasic sites, oxidized and alkylated bases, as well as mismatches arising from deamination of cytosine and 5-methylcytosine. Some of these lesions are repaired by the exchange of a single deoxynucleotide [Dianov, G. et al. (1992) Mol. Cell. Biol. 12, 1605-1612; Wiebauer, K. and Jiricny, J. (1990) Proc. Natl. Acad. Sci. USA, 87, 5842-5845] or a few deoxynucleotides [Matsumoto, Y. et al. (1994) Mol. Cell. Biol., 14 6187-6197]. Here we report that DNA single strand breaks induced by hyperthermic conditions are repaired with an average patch size of approximately 36 nt in Xenopus laevis egg lysates.     

The effects of estrogen, insulin and dexamethasone on the synthesis and secretion of egg white proteins in primary cultured oviduct cells of laying Japanese quail (Coturnix coturnix japonica)

1. The effects of estrogen, insulin and dexamethasone on the synthesis of egg white proteins were investigated by employing primary cultured oviduct cells of laying Japanese quails. 2. It was demonstrated that oviduct cells require insulin and dexamethasone, besides estrogen, to synthesize and secrete egg white proteins maximally.     

The Relationship of the Estrogen Receptor to the Induction of Vitellogenin in Chicken and Xenopus Liver

The mechanisms involved in the regulation of gene expression in eukaryote cells, although an area of active research, are still largely unknown. This is at least partly due to the lack of good experimental model systems. One type of system which is being exploited with some considerable success is the induction of proteins by steroid hormones. Studies on the effects of estrogen and progesterone on the synthesis of the egg white proteins in the chick oviduct, for instance, have yielded substantial insight into both the regulation of protein synthesis by steroid hormones [1] and the arrangement of the DNA sequences coding for these proteins [2, 3].
The need for other good inducible systems clearly exists and the induction of vitellogenin, the precursor of the major egg yolk proteins, by estrogen in the livers of the chicken and frog ( Xenopus laevis ) is one that is attracting increasing interest. In common with the chick oviduct, large amounts of a specific protein are synthesised in response to a well defined hormonal stimulus. However, the induction of vitellogenin also has the advantage that the response is not complicated by the extensive hyperplasia that follows estrogen treatment in the chick oviduct [4, 5] and that vitellogenin may be induced in vitro [6–11].
The aims of this review are first to discuss recent data on the induction of vitellogenin and vitellogenin mRNA both in vivo and in vitro and then to relate this data to the properties of the estrogen receptor, present in chicken and Xenopus liver, which is thought to mediate the induction of vitellogenin by estrogen.     

Estrogen Receptor Is Not Primarily Responsible for Altered Responsiveness of Ovalbumin mRNA Induction in the Oviduct From Genetically Selected High- and Low-Albumen Chicken Lines

The role of estrogen receptor on ovalbumin mRNA induction by steroid hormones was investigated in primary cultures of oviduct cells from estrogen-stimulated immature chicks of genetically selected high- and low-albumen egg laying lines (H- and L-lines). In experiment 1,the extent of ovalbumin mRNA induction and changes in estrogen and progesterone receptors were compared between the oviduct cells from H- and L-lines with or without steroid hormones in the culture medium. In experiment 2, the effect of estrogen receptor gene transfection on the induction of ovalbumin mRNA was studied in the oviduct cells from the L-line chicks. The results showed a close correlation of the changes in ovalbumin mRNA with the numbers of nuclear and total estrogen receptors in the oviduct cells but not with the numbers of nuclear and total progesterone receptors. Estrogen receptor gene transfection induced ovalbumin mRNA to a moderate extent in the absence of the steroid hormones. To our surprise, however, estrogen receptor gene transfection apparently suppressed the ovalbumin mRNA responsiveness to estrogen to a considerable extent. It was concluded, therefore, that the extent of estrogen receptor expression might not be primarily responsible for the differences in responsiveness to steroid hormones of oviduct cells from genetically selected H- and L-line chickens.     

Expression of glutathione transferase isoenzymes in the human H295R adrenal cell line and the effect of forskolin

In previous studies in our laboratory (L. Mankowitz, L. Staffas, M. Bakke, and J. Lund, Biochem J, 1995, 305, 111-118; L. Staffas, L. Mankowitz, M. S?derstr?m, A. Blanck, I. Porsch-H?llstr?m, C. Sundberg, B. Mannervik, B. Olin, J. Rydstr?m, and J.W. DePierre, Biochem J, 1992, 286, 65-72) isoenzymes of GST, primarily of the mu class, have been shown to be downregulated by adrenocorticotropic hormone (ACTH) in rat and mouse adrenal cells. In the present investigation the human adrenal H295R cell line (W.E. Rainey, I.M. Bird, and J.I. Mason, Mol Cell Endocrinol, 1994, 100, 45-50) was examined in a similar manner. Analysis by reverse-phase HPLC revealed that these cells express four isoenzymes of GST, i.e., A1, A2, P1, and M4, as well as another unidentified protein that was retained by our affinity column (elution time of 32 min) and, thus, presumably binds glutathione. Among these forms, A1 was present at the highest level. Upon addition of forskolin (an activator of adenylate cyclase which has been shown previously to mimic the effect of ACTH on adrenal cells) to the culture medium, the level of A1 decreased approximately 70% by forskolin, whereas the levels of the other isoenzymes were slightly increased, and that of the unknown form doubled. Thus, the influence of ACTH on expression of GST isoenzymes in this human adrenal cell line differs from that in rat and mouse adrenal cells.