Active mitochondria support osteogenic differentiation by stimulating ß-catenin acetylation.

Bone marrow stromal (a.k.a. mesenchymal stem) cells (BMSCs) can differentiate into osteoblasts (OBs), adipocytes, or chondrocytes. As BMSCs undergo OB differentiation, they up-regulate mitochondrial oxidative phosphorylation (OxPhos). Here, we investigated the mechanism(s) connecting mitochondrial OxPhos to OB differentiation. First, we found that treating BMSC-like C3H10T1/2 cells with an OxPhos inhibitor reduces their osteogenic potential. Interestingly, ATP levels were not reduced, as glycolysis compensated for the decreased OxPhos. Thus, mitochondria support OB differentiation not only by supplying ATP, but also by other mechanisms. To uncover these mechanisms, we stimulated OxPhos in C3H10T1/2 cells by replacing media glucose with galactose and observed that this substitution increases both OxPhos and osteogenesis even in the absence of osteoinducers. ß-Catenin, an important signaling pathway in osteogenesis, was found to be responsive to OxPhos stimulation. ß-Catenin activity is maintained by acetylation, and mitochondria generate the acetyl donor acetyl-CoA, which upon entering the Krebs cycle is converted to citrate capable of exiting mitochondria. Cytosolic citrate is converted back to acetyl-CoA by ATP citrate lyase (ACLY). We found that inhibiting ACLY with SB204990 (SB) reverses the galactose-induced ß-catenin activity and OB differentiation. This suggested that acetylation is involved in ß-catenin activation after forced OxPhos stimulation, and using immunoprecipitation, we indeed detected SB-sensitive ß-catenin acetylation. Both ß-catenin acetylation and activity increased during osteoinduction coincident with OxPhos activation. These findings suggest that active mitochondria support OB differentiation by promoting ß-catenin acetylation and thus activity.

Pancreatic ductal adenocarcinoma concomitant with pancreatic metastases of clear-cell renal cell carcinoma: a case report.

BACKGROUND: Metastatic spread to the pancreas is a rare event. Renal cell carcinoma represents one possible site of origin of pancreatic metastases. Renal cell carcinoma often metastasizes late and exclusively to the pancreas, suggesting a special role of renal cell carcinoma among primaries metastasizing to the pancreas. Even rarer, renal cell carcinoma may occur simultaneously with pancreatic ductal adenocarcinoma. CASE PRESENTATION: We present the case of a 78-year-old male Caucasian patient with a history of clear-cell renal cell carcinoma treated with oncological left nephrectomy 20 years before. The patient was diagnosed with pancreatic ductal adenocarcinoma by fine-needle aspiration cytology. At our institution, he received neoadjuvant therapy with folic acid, fluorouracil, irinotecan, oxaliplatin for borderline-resectable pancreatic ductal adenocarcinoma, and subsequently underwent total pancreatectomy. Upon resection, pancreatic ductal adenocarcinoma as well as two metachronous metastases of clear-cell renal cell carcinoma occurring simultaneously and cospatially with pancreatic ductal adenocarcinoma were diagnosed in the pancreatic body. CONCLUSIONS: Renal cell carcinoma metastases of the pancreas are rare and often occur decades after the initial diagnosis of renal cell carcinoma. The combination of renal cell carcinoma metastases and pancreatic ductal adenocarcinoma is even rarer. However, the possibility should be considered by clinicians, radiologists, and pathologists. The special role of renal cell carcinoma as a site of origin of pancreatic metastasis should be further elucidated.

Peripheral non-viral MIDGE vector-driven delivery of beta-endorphin in inflammatory pain.

BACKGROUND: Leukocytes infiltrating inflamed tissue produce and release opioid peptides such as beta-endorphin, which activate opioid receptors on peripheral terminals of sensory nerves resulting in analgesia. Gene therapy is an attractive strategy to enhance continuous production of endogenous opioids. However, classical viral and plasmid vectors for gene delivery are hampered by immunogenicity, recombination, oncogene activation, anti-bacterial antibody production or changes in physiological gene expression. Non-viral, non-plasmid minimalistic, immunologically defined gene expression (MIDGE) vectors may overcome these problems as they carry only elements needed for gene transfer. Here, we investigated the effects of a nuclear localization sequence (NLS)-coupled MIDGE encoding the beta-endorphin precursor proopiomelanocortin (POMC) on complete Freund's adjuvant-induced inflammatory pain in rats. RESULTS: POMC-MIDGE-NLS injected into inflamed paws appeared to be taken up by leukocytes resulting in higher concentrations of beta-endorphin in these cells. POMC-MIDGE-NLS treatment reversed enhanced mechanical sensitivity compared with control MIDGE-NLS. However, both effects were moderate, not always statistically significant or directly correlated with each other. Also, the anti-hyperalgesic actions could not be increased by enhancing beta-endorphin secretion or by modifying POMC-MIDGE-NLS to code for multiple copies of beta-endorphin. CONCLUSION: Although MIDGE vectors circumvent side-effects associated with classical viral and plasmid vectors, the current POMC-MIDGE-NLS did not result in reliable analgesic effectiveness in our pain model. This was possibly associated with insufficient and variable efficacy in transfection and/or beta-endorphin production. Our data point at the importance of the reproducibility of gene therapy strategies for the control of chronic pain.

Lymphocytes upregulate signal sequence-encoding proopiomelanocortin mRNA and beta-endorphin during painful inflammation in vivo.

Proopiomelanocortin (POMC)-derived beta-endorphin1-31 (END) released from immune cells inhibits inflammatory pain. We examined the expression of END and POMC mRNA encoding the signal sequence required for entry of the nascent polypeptide into the regulated secretory pathway in lymphocytes of rats with inflamed hindpaws. Within 12 h of inflammation, END increased in popliteal lymph nodes and at 96 h the intraplantar neutralization of END exacerbated pain. Lymphocytes expressed POMC, END, and full-length POMC mRNA. Semi-nested PCR revealed 8-fold increased exon 2-3 spanning POMC mRNA. Thus, painful inflammation enhances signal sequence-encoding lymphocytic POMC mRNA needed for regulated secretion of functionally active END.