Gene Expression and the Physiological Role of Transforming Growth Factor-α in the Mouse Pituitary

Transforming growth factor-alpha (TGF-alpha), a member of the epidermal growth factor (EGF) family, is produced within the mouse anterior pituitaries. However, the cell types of TGF-alpha-expressing cells and the physiological roles of TGF-a within mouse pituitary glands remain unclear. The aim of the present study was to localize TGF-alpha mRNA-expressing cells, and to clarify the involvement of TGF-alpha in estrogen-induced DNA replication in mouse anterior pituitary cells. Northern blot analysis demonstrated TGF-alpha mRNA expression in adult male and female mouse anterior pituitaries. In situ hybridization analysis of the pituitaries in these mice showed that TGF-alpha mRNA-expressing cells in the anterior pituitary are round, oval, and medium-sized. TGF-alpha mRNA was colocalized in most of the growth hormone (GH) mRNA-expressing cells, while only some of the prolactin (PRL) mRNA-expressing cells. DNA replication in the anterior pituitary cells was detected by monitoring the cellular uptake of a thymidine analogue, bromodeoxyuridine (BrdU) in a primary serum-free culture system. Estradiol-17beta (E2) and TGF-alpha treatment increased the number of BrdU-labelled mammotrophs, indicating that E2 and TGF-alpha treatment stimulates the DNA replication in mammotrophs. Immunoneutralization of TGF-alpha with anti-TGF-alpha-antibodies nullified the E2-induced increase in DNA replication. RT-PCR analysis of TGF-alpha mRNA expression in ovariectomized female mice revealed that E2 increases TGF-alpha mRNA levels. These results indicate that the TGF-alpha produced primarily in the somatotrophs mediates the stimulatory effects of estrogen on the DNA replication of pituitary cells in a paracrine or autocrine manner

Embryonic dermal condensation and adult dermal papilla induce hair follicles in adult glabrous epidermis through different mechanisms

Hair induction in the adult glabrous epidermis by the embryonic dermis was compared with that by the adult dermis. Recombinant skin, composed of the adult sole epidermis and the embryonic dermis containing dermal condensations (DC), was transplanted onto the back of nude mice. The epidermis of transplants formed hairs. Histology on the induction process demonstrated the formation of placode-like tissues, indicating that the transplant produces hair follicles through a mechanism similar to that underlying hair follicle development in the embryonic skin. An isolated adult rat sole skin piece, inserted with either an aggregate of cultured dermal papilla (DP) cells or an intact DP between its epidermis and dermis, was similarly transplanted. The transplant produced hair follicles. Histology showed that the epidermis in both cases surrounded the aggregates of DP cells. The epidermis never formed placode-like tissues. Thus, it was concluded that the adult epidermal cells recapitulate the embryonic process of hair follicle development when exposed to DC, whereas they get directly into the anagen of the hair cycle when exposed to DP. The expression pattern of Edar and Shh genes, and P-cadherin protein during the hair follicle development in the two types of transplants supported the above conclusion

The extract of syngeneic keratinocytes enhances IgE production from BALB/c mouse splenic lymphocytes in vitro.

Background: The increase of serum IgE levels is closely associated with atopic dermatitis. We have previously revealed that cellular extract of PAM212 cells (PAM-extract), BALB/c mouse keratinocyte cell line, induced a remarkable increase of serum IgE levels, in vivo, when subcutaneously injected into BALB/c mice. However, precise mechanism of IgE-increasing activity was unclear. Objective: To elucidate the mechanism of IgE-increase in sera of BALB/c mice induced by PAM-extract, we explored the direct influence of PAM-extract on immunoglobulin production and class-switching in the culture of splenic lymphocytes and purified B-cells, in vitro. Methods: Splenic lymphocytes or purified B-cells obtained from BALB/c mice were cultured with various combinations of IL-4, anti-CD40 antibody, and PAM-extract for seven days. IgE and IgG concentrations of culture supernatants were measured by ELISA. Epsilon germ-line transcriptions were assessed by RT-PCR from the cultured cells. Results: IgE and IgG concentrations in culture supernatant of splenic lymphocytes were increased by an addition of PAM-extract in the presence of both IL-4 and anti-CD40 antibody. Epsilon germ-line transcript was also induced in parallel to the increase of IgE production. Similar results were obtained when purified B-cells were employed in stead of whole splenic lymphocytes. Conclusion: The cellular extract of keratinocyte promotes immunoglobulin class-switching to IgE and IgE production from mouse splenic B-cells in an IL-4- and CD40-stimuli-dependent manner. Such enhancement may account for the increase of serum IgE in patients with dermatitis in association with a Th2 microenvironment

Proliferation of Pituitary Cells in Streptozotocin-induced Diabetic Mice : Effect of Insulin and Estrogen

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Peritoneal injection of fucoidan suppresses the increase of plasma IgE induced by OVA-sensitization

We previously reported that fucoidan, a dietary fiber purified from seaweed, inhibited IgE production by B cells in vitro. In this study, we examined the effect of fucoidan on IgE production in vivo. The OVA-induced increase of plasma IgE was significantly suppressed when fucoidan was intraperitoneally, but not orally, administered prior to the first immunization with OVA. The production of IL-4 and IFN-gamma in response to OVA in spleen cells isolated from OVA-sensitized mice treated with fucoidan in vivo was lower than that from mice treated without fucoidan. Moreover, the flow cytometric analysis and ELISpot assay revealed that the administration of fucoidan Suppressed a number of IgE-expressing and IgE-secreting B cells, respectively. These results indicate that fucoidan inhibits the increase of plasma IgE through the suppression of IgE-producing B cell population, and the effect of fucoidan in VIVO is Crucially dependent on the route and timing of its administration

Expansion of Gal-9⁺ Th cells by exogenous Gal-9.

<p>(<b>A</b>) Naïve CD4 T cells were cultured under unstimulated, neutral, or Th17-skewing conditions for 4 days in the presence or absence of 30 nM human stable Gal-9 before surface Gal-9 expression was monitored by flow cytometry using an anti-mouse Gal-9 antibody. The antibody does not cross-react with the added human Gal-9. Dot plots are representative results obtained from neutral conditions in the presence or absence of exogenous Gal-9. (<b>B and C</b>) Flow cytometric analysis of Gal-9⁺ Th cell frequency after 4-day culture of naïve CD4 T cells under neutral conditions in the presence of blocking anti-IL-10 or anti-TGF-β antibody (<b>B</b>) or in the presence of the indicated concentration of IL-10 or 30 nM human stable Gal-9 (<b>C</b>). Results are means ± SEMs of quadruplicates. <i>p</i><0.001 (***), <i>p</i><0.01 (**), <i>p</i><0.05 (*), not significant (NS). Representative data out of 2 experiments are shown.</p

Identification of Gal-9⁺ Th cells.

<p>(<b>A</b>) Naïve CD4 T cells were cultured as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048574#pone-0048574-g001" target="_blank"><b>Figure 1A</b></a>, and cell-surface Gal-9 expression was monitored using flow cytometry. (<b>B</b>) Naïve CD4 T cells were sorted into Gal-9⁺ Th and Gal-9⁻ Th cells with a cell sorter and cultured under TCR stimulation or left unstimulated for 4 days before Gal-9 secretion into the culture media was measured. (<b>C</b>) Gal-9⁺ Th and Gal-9⁻ Th cells cultured under TCR stimulation for 4 days were examined for Gal-9 mRNA expression (left) and intracellular Gal-9 protein expression (right). (<b>D</b>) Cytokine mRNA expression in Gal-9⁺ Th and Gal-9⁻ Th cells cultured with or without TCR stimulation for 4 days. All the results are shown as the mean ± SEM of quadruplicates. <i>p</i><0.001 (***), <i>p</i><0.05 (*), not significant (NS). Representative data out of at least 2 experiments are shown.</p