Mark A. Kessler

Regulation of interleukin (IL)-1α, IL-1β, and IL-6 expression by growth hormone and prolactin in bovine thymic stromal cells

Abstract Growth hormone (GH) and prolactin (PRL) have been implicated in T-cell development, but relatively little is known about the mechanism(s) of their actions on the multiple cell types in this complex tissue. Here, we investigated the effects of GH and PRL on the expression of interleukin (IL)-1α, IL-1β and IL-6 in thymic stromal cells (TSC). These cytokine mRNAs were increased by GH, PRL and placental lactogen (PL) in primary cultures prepared from mid-gestational fetuses in a dose-dependent manner. IL-1 receptor antagonist (IL-1ra) abolished the hormone-induced IL-6 expression, suggesting that the induction of IL-6 was secondary to IL-1 activity. To examine the effects of these hormones on an individual cell type and develop a system in which signalling mechanisms can be studied, we generated immortalized cell lines using a strategy of conditional transformation. In the cell line, TSC-936, which displayed vimentin-positive staining and morphological characteristics of mesenchymal cells, both GH and PRL increased levels of steady-state mRNAs for IL-1α and IL-1β. Nuclear run-on analysis revealed that the transcription rate of the IL-1β gene was significantly increased by GH and PRL at 30 and 60 min, respectively, but that for IL-1α was not significantly changed, suggesting the possibility of an alternative mechanism mediating this response. These data suggest that modulation of cytokine gene expression is one mechanism by which GH and PRL facilitate thymic development and T-cell maturation.

Bovine placental prolactin-related hormones

The bovine placenta, like that of rodents and primates, synthesizes members of the PRL/GH gene family, which may assist the pituitary hormones or perform unique functions during pregnancy. Bovine placental lactogen (bPL) potentially may act through.three receptors: as an agonist or partial antagonist at the PRL and GH receptors, and via an apparently specific receptor in the endometrium. A large distinct subfamily of diverse primary structure, including bovine PRL-related protein I (bPRP-I), evidently does not act via these receptors. Advances in our understanding of hormone-receptor interactions for this gene family have provided new tools to study the role of these hormones in the successful pregnancy.

1,25-Dihydroxyvitamin D3-stimulated mRNAs in rat small intestine☆

The technique of differential hybridization has been employed to study gene expression associated with vitamin D action on the mammalian intestine. A cDNA library consisting of 10(6) independent recombinants was constructed from poly(A)+ RNA extracted from vitamin D-deficient rats given 1,25-dihydroxyvitamin D3. A survey of 20,000 clones resulted in identification of four distinct cDNAs whose corresponding mRNAs are significantly increased 12 h after an intrajugular dose of 1,25-dihydroxyvitamin D3 given to vitamin D-deficient rats. DNA sequence analysis identified these mRNAs as mitochondrial ATP synthetase, vitamin D-dependent calcium binding protein, cytochrome oxidase subunit I, and cytochrome oxidase subunit III. The time course of response of three of these mRNAs was similar, with maximum values at 12 h after dosing, while that of cytochrome oxidase subunit I showed two peaks at 6 and 18 h following a single dose of 1,25-dihydroxyvitamin D3. The levels of all four mRNAs were elevated in rats supplied with vitamin D when hypocalcemia was produced by dietary calcium restriction.

SmMAK16, the Schistosoma mansoni homologue of MAK16 from yeast, targets protein transport to the nucleolus.

The SmMAK16 gene from Schistosoma mansoni was cloned by chance when an adult worm cDNA library was probed with antiserum to affinity-purified S. mansoni GSH S-transferases. SmMAK16 encodes a hydrophilic protein of 259 amino acids with a molecular mass of 31 kDa. The protein shares 43% sequence identity and 66% similarity to the nuclear protein MAK16 of Saccharomyces cerevisiae that has been implicated both in cell cycle progression and biogenesis of 60S ribosomal subunits. Both proteins display a similar degree of sequence similar to the hypothetical protein CeMAK16 from Caenorhabditis elegans. These proteins share a number of apparent protein motifs, including two nuclear localization signals (NLS), multiple sites for phosphorylation by protein kinase CK2 and four conserved cysteine residues that resemble a zinc binding domain. SmMAK16 mRNA is more highly expressed in adult female worm than males. Recombinant SmMAK16 was phosphorylated by human protein kinase CK2. When chimeric constructs containing SmMAK16 fused the green fluorescent protein (GFP) were transiently transfected into COS-7s cells, the reporter was localized not in nuclei, but exclusively in nucleoli. The yeast and nematode homologues were likewise able to direct nucleolar accumulation of the fluorescent reporter. The high degree of sequence conservation together with the ability to direct nucleolar protein transport supports the hypothesis that MAK16 proteins play a key role in the biogenesis of 60S subunits.

The action of 1 α,25-dihydroxyvitamin D3 on rat small intestine: Changes in translatable mRNA species☆

The time course of changes in translatable mRNA from small intestinal mucosa was studied in vitamin D-deficient rats following an intrajugular dose of 1,25-dihydroxyvitamin D3. L-[35S]Methionine-labeled translation products were analyzed by high-resolution one- and two-dimensional electrophoresis. Changes were detected in two distinct proteins, Mr 14,000/pI 6.24 (Band I), and Mr 5,200/pI 4.86 (Band II). These increased to maxima of 1-2% each of the total in vitro-synthesized protein at 12 h after dosing. This represented a 3- and 10-fold increase, respectively, over control values. The time course of these changes suggests that regulation of the levels of the mRNAs coding for these proteins is not involved in the initial (6 h) peak of 1,25-dihydroxyvitamin D3-stimulated intestinal calcium transport in the rat.

Molecular Genetics and Biology of the Bovine Placental Prolactin Family

The placenta produces many factors and hormones that are believed to help coordinate the successful pregnancy, including members of theprolactin (PRL)/growth hormone (GH) gene family. Despite considerable conjecture about the function of these placental PRL-GH gene family members based on analogy to GH and PRL, little is known about the actions of these hormones. The complex coordination of fetal development and maternal adaptations to pregnancy and the breadth of activities modulated by GH and PRL suggest a wide range of potential targets within the fetus, mother, and uteroplacental unit. It has been suggested that these hormones may be involved in fetal and/or placental growth, modulation of maternal energy metabolism, growth of the mammary epithelium, maintenance of steroidogenesis by both the placenta and corpus luteum, control of osmotic balance in the fetus, development of fetal immune competence, and modulation of the maternal immune system. However, species differences in placental anatomy and the biology of pregnancy itself have proven generalizations risky. Even in those species where placentally expressed relatives of the pituitary hormones have been identified, distinct primary amino acid sequences, secondary structure, and patterns of expression have rendered experiments employing heterologous systems nearly uninterpretable.