Pentraxin 3 (PTX3) is a prototypic humoral soluble pattern-recognition molecule known to function in immunity-related inflammation. Given the lack of information on the precise functions of PTX3 in the pathogenesis of Graves’ orbitopathy (GO), this study investigated the role of PTX3 in the inflammation and adipogenesis mechanism of GO. We first compared the PTX3 expression between orbital tissues from patients with GO and normal controls, using real-time polymerase chain reaction, which estimated significantly higher PTX3 transcript levels in the GO tissues than in the normal tissues. In addition, PTX3 production was markedly increased upon interleukin (IL)-1β and adipogenic stimulation. We then evaluated the effects of silencing PTX3 in primary orbital fibroblast cultures by analyzing the expression levels of pro-inflammatory cytokines, adipogenesis-related proteins, and downstream transcription factors in cells transfected with or without a small interfering RNA against PTX3, using western blot. Silencing PTX3 attenuated the IL-1β-induced secretion of pro-inflammatory cytokines, including IL-6, IL-8, monocyte chemotactic protein-1, intercellular adhesion molecule-1, and cyclooxygenase-2, and suppressed the IL-1β-mediated activation of p38 kinase, nuclear factor-κB, and extracellular signal-regulated kinase. Moreover, PTX3 knockdown suppressed adipogenic differentiation, as assessed using Oil Red O staining, as well as the expression of adipogenesis-associated transcription factors including peroxisome proliferator activator-γ, CCAAT/enhancer-binding proteins α and β, adipocyte protein 2, adiponectin, and leptin. Thus, this study suggests that PTX3 plays a significant role in the pathogenesis of GO and may serve as a novel therapeutic target for the condition.

Androgens act in concert with vitamin D to influence reabsorption of calcium. However, it is unclear whether androgens directly regulate vitamin D homeostasis or control other cellular events that are related to vitamin D metabolism. To examine whether the expression of vitamin D-related genes in mouse kidney is driven by androgens or androgen-dependent effects, the androgen receptor and other sex steroid receptors were monitored in orchidectomized mice treated with 5α-dihydrotestosterone (DHT). Our results revealed that exposing orchidectomized mice to DHT inhibited the expression of progesterone receptor (Pgr) with or without estrogen receptor α expression, the latter was confirmed by ER-positive (MCF7 and T47D) or -negative (PCT) cells analysis. The loss of Pgr in turn decreased the expression of renal 24-hydroxylase via transcriptional regulation because Cyp24a1 gene has a progesterone receptor-binding site on promoter. When male kidneys preferentially hydroxylate 25-hydroxyvitamin D₃ using 24-hydroxylase rather than 25-hydroxyvitamin D₃-1-alpha hydroxylase, DHT suppressed the Pgr-mediated 24-hydroxylase expression, and it is important to note that DHT increased the blood 25-hydroxyvitamin D₃ levels. These findings uncover an important link between androgens and vitamin D homeostasis and suggest that therapeutic modulation of Pgr may be used to treat vitamin D deficiency and related disorders.

Angiotensin II (Ang-II) and vascular endothelial growth factor (VEGF) have an important role in the pathogenesis of diabetic nephropathy, but the signaling cascade of VEGF regulation in response to Ang-II in podocytes is largely unknown. In these experiments, we looked at the effect of Ang-II on the production of VEGF, and investigated whether VEGF production depends on the p38 mitogen activated protein kinase (MAPK) pathway in cultured mouse podocytes. Incubation of podocytes with Ang-II induced a rapid increase in VEGF mRNA expression and protein synthesis as well as its transcriptional activity in an Ang-II dose-dependent manner. To further define the role of angiotensin type 1 (AT1) and type 2 (AT2) receptors involved in Ang-II-mediated VEGF synthesis, the effects of selective AT1 and AT2 receptor antagonists were evaluated. Prior treatment with losartan significantly inhibited VEGF mRNA and protein synthesis induced by Ang-II, which suggests that the AT1 receptor is involved in Ang-II-mediated VEGF synthesis. Furthermore, stimulation of the cells with Ang-II increased both phosphorylation of p38 MAPK and MAP kinase kinase 3/6 (MKK3/6). Additionally, Ang-II enhanced the DNA binding activity to cAMP response element binding protein (CREB) and phosphorylation of CREB. In addition, to investigate the role of p38 MAPK in Ang-II-induced VEGF synthesis, podocytes were pretreated with or without the p38 MAPK inhibitor, SB203580 for 24 h to observe whether Ang-II-mediated VEGF synthesis was inhibited by blocking p38 MAPK. The addition of SB203580 led to a marked inhibition of the increased VEGF mRNA and protein production induced by Ang-II in a dose-dependent manner. Taken together, these results suggest that Ang-II stimulates the synthesis of VEGF in podocytes and the production of VEGF induced by Ang-II is mediated, in part, through the activation of the p38 MAPK pathway.