Modulation of adenine phosphoribosyltransferase-mediated salvage pathway to accelerate diabetic wound healing.

Adenine phosphoribosyltransferase (APRT) is the key enzyme involved in purine salvage by the incorporation of adenine and phosphoribosyl pyrophosphate to provide adenylate nucleotides. To evaluate the role of APRT in the repair processes of cutaneous wounds in healthy skin and in diabetic patients, a diabetic mouse model (db/db) and age-matched wild-type mice were used. Moreover, the topical application of adenine was assessed. In vitro studies, analytical, histological, and immunohistochemical methods were used. Diabetic mice treated with adenine exhibited elevated ATP levels in organismic skin and accelerated wound healing. In vitro studies showed that APRT utilized adenine to rescue cellular ATP levels and proliferation from hydrogen peroxide-induced oxidative damage. HPLC-ESI-MS/MS-based analysis of total adenylate nucleotides in NIH-3T3 fibroblasts demonstrated that adenine addition enlarged the cellular adenylate pool, reduced the adenylate energy charge, and provided additional AMP for the further generation of ATP. These data indicate an upregulation of APRT in skin wounds, highlighting its role during the healing of diabetic wounds through regulation of the nucleotide pool after injury. Furthermore, topical adenine supplementation resulted in an enlargement of the adenylate pool needed for the generation of ATP, an important molecule for wound repair.

The anti-inflammatory function of adenine occurs through AMPK activation and its downstream transcriptional regulation in THP-1 cells.

Pathogenic bacteria induced sepsis is a risk factor for hospital mortality. Monocyte-derived inflammatory cytokines participate in the sepsis progression. The anti-inflammatory effect of adenine has been previously reported by our laboratory and others. However, the mechanism of action has different opinions and remains unclear in monocyte. Here, adenine was found to significantly inhibit the secretion of lipopolysaccharide-induced inflammatory cytokines such as TNF-α, IL-1ß and IL-6 in THP-1 cells. The bioinformatic analysis results showed that the anti-inflammatory function is possibly due to the inhibition of NF-κB signaling. And this result is confirmed by using immunocytochemistry. Moreover, this effect can be suppressed by the AMPK inhibitor. Results also showed that adenine can activate AMPK and its multiple downstream targets. Data from mass spectrometry showed that adenine promotes significant elevation of intracellular AMP. Our data indicate that the anti-inflammatory mechanism of adenine may involve adenine phosphoribosyltransferase-catalyzed intracellular AMP elevation, which stimulates AMPK activation.

Activation of AMP-Activated Protein Kinase by Adenine Alleviates TNF-Alpha-Induced Inflammation in Human Umbilical Vein Endothelial Cells.

The AMP-activated protein kinase (AMPK) signaling system plays a key role in cellular stress by repressing the inflammatory responses induced by the nuclear factor-kappa B (NF-κB) system. Previous studies suggest that the anti-inflammatory role of AMPK involves activation by adenine, but the mechanism that allows adenine to produce these effects has not yet been elucidated. In human umbilical vein endothelial cells (HUVECs), adenine was observed to induce the phosphorylation of AMPK in both a time- and dose-dependent manner as well as its downstream target acetyl Co-A carboxylase (ACC). Adenine also attenuated NF-κB targeting of gene expression in a dose-dependent manner and decreased monocyte adhesion to HUVECs following tumor necrosis factor (TNF-α) treatment. The short hairpin RNA (shRNA) against AMPK α1 in HUVECs attenuated the adenine-induced inhibition of NF-κB activation in response to TNF-α, thereby suggesting that the anti-inflammatory role of adenine is mediated by AMPK. Following the knockdown of adenosyl phosphoribosyl transferase (APRT) in HUVECs, adenine supplementation failed to induce the phosphorylation of AMPK and ACC. Similarly, the expression of a shRNA against APRT nullified the anti-inflammatory effects of adenine in HUVECs. These results suggested that the role of adenine as an AMPK activator is related to catabolism by APRT, which increases the cellular AMP levels to activate AMPK.

Amelioration of LPS-induced inflammation response in microglia by AMPK activation.

Adenosine 5'-monophosphate-activated protein kinase (AMPK) is a key regulator of cellular energy homeostasis via modulating metabolism of glucose, lipid, and protein. In addition to energy modulation, AMPK has been demonstrated to associate with several important cellular events including inflammation. The results showed that ENERGI-F704 identified from bamboo shoot extract was nontoxic in concentrations up to 80 µM and dose-dependently induced phosphorylation of AMPK (Thr-172) in microglia BV2 cells. Our findings also showed that the treatment of BV2 with ENERGI-F704 ameliorated the LPS-induced elevation of IL-6 and TNF-α production. In addition, ENERGI-F704 reduced increased production of nitric oxide (NO) and prostaglandin E2 (PGE2) via downregulating the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2), respectively. Moreover, ENERGI-F704 decreased activated nuclear translocation and protein level of NF-κB. Inhibition of AMPK with compound C restored decreased NF-κB translocation by ENERGI-F704. In conclusion, ENERGI-F704 exerts inhibitory activity on LPS-induced inflammation through manipulating AMPK signaling and exhibits a potential therapeutic agent for neuroinflammatory disease.

Progesterone receptor is involved in 2,3,7,8-tetrachlorodibenzo-p-dioxin-stimulated breast cancer cells proliferation.

Sex hormones are pivotal for both normal and neoplastic development of breast tissues. TCDD, with sex hormonal activity, executes multiple biological activities primarily through AhR. However, the detailed mechanisms how TCDD affects human breast cell are mostly unexplored. We analyzed the biological effects and underlying mechanisms of TCDD on MCF-7 cells. PR, other than AhR, was involved in TCDD-stimulated MCF-7 cell proliferation. TCDD inactivated Akt-FoxO3a pathway, increased cdc25C/cdc2 activity, decreased P21/P27 activity, and enriched G2/M phase, in a PR- and AhR-dependent manner. In conclusion, our study demonstrated for the first time that PR was involved in TCDD-stimulated breast cell proliferation.