Inhibition of Phosphodiesterase 5 Promotes the Aromatase-Mediated Estrogen Biosynthesis in Osteoblastic Cells by Activation of cGMP/PKG/SHP2 Pathway.

Mechanical stimulation induces bone growth and remodeling by the secondary messenger, cyclic guanosine 3', 5'-monophosphate (cGMP), in osteoblasts. However, the role of cGMP in the regulation of estrogen biosynthesis, whose deficiency is a major cause of osteoporosis, remains unclear. Here, we found that the prenylated flavonoids, 3-O-methoxymethyl-7-O-benzylicaritin (13), 7-O-benzylicaritin (14), and 4'-O-methyl-8-isopentylkaempferol (15), which were synthesized using icariin analogs, promoted estrogen biosynthesis in osteoblastic UMR106 cells, with calculated EC50 values of 1.53, 3.45, and 10.57 µM, respectively. 14 and 15 increased the expression level of the bone specific promoter I.4-driven aromatase, the only enzyme that catalyzes estrogen formation by using androgens as substrates, in osteoblastic cells. 14 inhibited phosphodiesterase 5 (PDE5), stimulated intracellular cGMP level and promoted osteoblast cell differentiation. Inhibition of cGMP dependent-protein kinase G (PKG) abolished the stimulatory effect of 14 on estrogen biosynthesis and osteoblast cell differentiation. Further, PKG activation by 14 stimulated the activity of SHP2 (Src homology 2 domain-containing tyrosine phosphatase 2), thereby activating Src and ERK (extracellular signal-regulated kinase) signaling and increasing ERK-dependent aromatase expression in osteoblasts. Our findings reveal a previously unknown role of cGMP in the regulation of estrogen biosynthesis in the bone. These results support the further development of 14 as a PKG-activating drug to mimic the anabolic effects of mechanical stimulation of bone in the treatment of osteoporosis.

Natural Polyketides Isolated from the Endophytic Fungus Phomopsis sp. CAM212 with a Semisynthetic Derivative Downregulating the ERK/IκBα Signaling Pathways.

Three previously undescribed natural products, phomopsinin A - C (1:  - 3: ), together with three known compounds, namely, cis-hydroxymellein (4: ), phomoxanthone A (5: ) and cytochalasin L-696,474 (6: ), were isolated from the solid culture of Phomopsis sp. CAM212, an endophytic fungus obtained from Garcinia xanthochymus. Their structures were determined on the basis of spectroscopic data, including IR, NMR, and MS. The absolute configurations of 1: and 2: were assigned by comparing their experimental and calculated ECD spectra. Acetylation of compound 1: yielded 1A: , a new natural product derivative that was tested together with other isolated compounds on lipopolysaccharide-stimulated RAW 264.7 cells. Cytochalasin L-696,474 (6: ) was found to significantly inhibit nitric oxide production, but was highly cytotoxic to the treated cells, whereas compound 1: slightly inhibited nitric oxide production, which was not significantly different compared to lipopolysaccharide-treated cells. Remarkably, the acetylated derivative of 1: , compound 1A: , significantly inhibited nitric oxide production with an IC50 value of 14.8 µM and no cytotoxic effect on treated cells, thereby showing the importance of the acetyl group in the anti-inflammatory activity of 1A: . The study of the mechanism of action revealed that 1A: decreases the expression of inducible nitric oxide synthase, cyclooxygenase 2, and proinflammatory cytokine IL-6 without an effect on IL-1ß expression. Moreover, it was found that 1A: exerts its anti-inflammatory activity in lipopolysaccharide-stimulated RAW 264.7 macrophage cells by downregulating the activation of ERK1/2 and by preventing the translocation of nuclear factor κB. Thus, derivatives of phomopsinin A (1: ), such as compound 1A: , could provide new anti-inflammatory leads.

Azaphilone Alkaloids with Anti-inflammatory Activity from Fungus Penicillium sclerotiorum cib-411.

Nine new azaphilone alkaloids, penazaphilones A-I (1-9), were isolated from the solid fermented rice culture of Penicillium sclerotiorum cib-411. The structures of compounds 1-9 were elucidated based on HRESIMS, NMR, and CD spectroscopic data. The structures of 5 and 8 were confirmed by X-ray crystallographic analyses. Biological evaluation showed that compounds 1, 5, 6, and 8 inhibited the production of nitric oxide (NO) on RAW 264.7 cells stimulated by lipopolysaccharide with IC50 values of 15.29, 9.34, 9.50, and 7.05 µM, respectively. Meanwhile, they did not exhibit obvious cytotoxicity at a concentration of 50.0 µM.

Inhibition of TPL2 by interferon-α suppresses bladder cancer through activation of PDE4D.

BACKGROUND: Drugs that inhibit the MEK/ERK pathway have therapeutic benefit in bladder cancer treatment but responses vary with patients, for reasons that are still not very clear. Interferon-α (IFN-α) is also used as a therapeutic agent for bladder cancer treatment but the response rate is low. It was found that IFN-α could enhance the cytotoxic effect of MEK inhibition. However, the potential mechanisms of that are still unclear. Understanding of the cross-talk between the IFN-α and MEK/ERK pathway will help enhance the efficacy of IFN-α or MEK inhibitors on bladder cancer. METHODS: Immunoprecipitation and pull-down assay were used to reveal the formation of signaling complex. The protein expressions were detected by western blot and immunohistochemistry. The cAMP level, Phosphodiesterase 4D (PDE4D) activity and Prostaglandin E2 (PGE2) concentration in cells, serum and tissues were detected by enzyme-linked immunosorbent assay. The role of PDE4D in bladder tumorigenesis in vivo was examined by the xenograft model. Tissue microarray chips were used to investigate the prognostic roles of PDE4D and tumor progression locus 2 (TPL2) in bladder cancer patients. RESULTS: IFN-α down-regulated the cyclooxygenase-2 (COX-2) expression in bladder cancer cells through the inhibition of TPL2/NF-κB pathway; IFN-α also inhibited COX-2 expression by suppressing cAMP signaling through TPL2-ERK mediated PDE4D activity. Reduction of the intracellular cAMP level by PDE4D potentiated the antitumor effect of IFN-α against bladder cancer in vitro and in vivo. Further analysis of clinical samples indicated that low PDE4D expression and high level of TPL2 phosphorylation were correlated to the development and poor prognosis in bladder cancer patients. CONCLUSIONS: Our data reveal that IFN-α can exert its antitumor effect through a non-canonical JAK-STAT pathway in the bladder cancer cells with low activity of IFN pathway, and the TPL2 inhibition is another function of IFN-α in the context of bladder cancer therapy. The antitumor effects of IFN-α and MEK inhibition also depend on the PDE4D-mediated cAMP level in bladder cancer cells. Suppression of the TPL2 phosphorylation and intracellular cAMP level may be possible therapeutic strategies for enhancing the effectiveness of IFN-α and MEK inhibitors in bladder cancer treatment.

[Double-ambient CO2 concentration affects the growth, development and sucking behavior of non-target brown plant hopper Nilaparvata lugens fed on transgenic Bt rice.] / CO2浓度倍增影响转Btæ°´ç¨»éžé¶æ ‡å®³è™«è¤é£žè™±çš„ç”Ÿé•¿å‘è‚²åŠå–é£Ÿè¡Œä¸º.

In recent years, the two issues of climate change including elevated CO2 etc., and resistance of transgenic Bt crops against non-target insect pests have received widespread attention. Elevated CO2 can affect the herbivorous insects. To date, there is no consensus about the effect of elevated CO2 on the suck-feeding insect pests (non-target insect pests of transgenic Bt crops). Its effects on the suck-feeding behavior have rarely been reported. In this study, CO2 levels were set up in artificial climate chamber to examined the effects of ambient (400 µL·L-1) and double-ambient (800 µL·L-1) CO2 levels on the suck-feeding behavior, growth, development, and reproduction of the non-target insect pest of transgenic Bt rice, brown planthopper, Nilaparvata lugens. The results showed that CO2 level significantly affected the egg and nymph duration, longevity and body mass of adults, and feeding behavior of the 4th and 5th instar nymphs, while had no effect on the fecundity of N. lugens. The duration of eggs and nymphs, and the longevity of female adults were significantly shortened by 4.0%, 4.2% and 6.6% respectively, the proportion of the macropterous adults was significantly increased by 11.6%, and the body mass of newly hatched female adults was significantly decreased by 2.2% by elevated CO2. In addition, elevated CO2 significantly enhanced the stylet puncturing efficiency of the 4th and 5th instar nymphs of N. lugens. The duration ofphloem ingestion of the N4b waveform was significantly prolonged by 60.0% and 50.1%, and the frequency significantly was increased by 230.0% and 155.9% for the 4th and 5th instar nymphs of N. lugens by elevated CO2, respectively. It was concluded that double-ambient CO2 could promote the growth and development of N. lugens through enhancing its suck-feeding, shorten the generation life-span and increase the macropertous adults' proportion of N. lugens. Thus, it could result in the occurrence of non-target rice planthopper N. lugens and make the transgenic Bt crops face with harm risk due to migration and diffusion of N. lugens under elevated CO2.