Intermittent administration of PTH stimulates bone formation, but the precise mechanisms responsible for PTH responses in osteoblasts are only incompletely understood. Here we show that binding of PTH to its receptor PTH1R induced association of LRP6, a coreceptor of Wnt, with PTH1R. The formation of the ternary complex containing PTH, PTH1R, and LRP6 promoted rapid phosphorylation of LRP6, which resulted in the recruitment of axin to LRP6, and stabilization of ␤-catenin. Activation of PKA is essential for PTH-induced ␤-catenin stabilization, but not for Wnt signaling. In vivo studies confirmed that PTH treatment led to phosphorylation of LRP6 and an increase in amount of ␤-catenin in osteoblasts with a concurrent increase in bone formation in rat. Thus, LRP6 coreceptor is a key element of the PTH signaling that regulates osteoblast activity.[Keywords: PTH signaling; LRP6; osteoblasts; ␤-catenin; PKA] Supplemental material is available at http://www.genesdev.org.

Parathyroid hormone (PTH) stimulates bone remodeling and induces differentiation of bone marrow mesenchymal stromal/stem cells (MSCs) by orchestrating activities of local factors such as bone morphogenetic proteins (BMPs). The activity and specificity of different BMP ligands are controlled by various extracellular antagonists that prevent binding of BMPs to their receptors. Low-density lipoprotein receptor-related protein 6 (LRP6) has been shown to interact with both the PTH and BMP extracellular signaling pathways by forming a complex with PTH1R and sharing common antagonists with BMPs. We hypothesized that PTH-enhanced differentiation of MSCs into the osteoblast lineage through enhancement of BMP signaling occurs by modifying the extracellular antagonist network via LRP6. In vitro studies using multiple cell lines, including Sca-1+CD45−CD11b− MSCs showed that a single injection of PTH enhanced phosphorylation of Smad1 and could also antagonize the inhibitory effect of noggin. PTH treatment induced endocytosis of a PTH1R/LRP6 complex and resulted in enhancement of phosphorylation of Smad1 that was abrogated by deletion of PTH1R, β-arrestin, or chlorpromazine. Deletion of LRP6 alone lead to enhancement of pSmad1 levels that could not be further increased with PTH treatment. Finally, knockdown of LRP6 increased the exposure of endogenous cell-surface BMPRII significantly in C2C12 cells and PTH treatment significantly enhanced cell surface binding of 125I-BMP2 in a dose- and time-dependent manner, implying that LRP6 organizes an extracellular network of BMP antagonists that prevent access of BMPs to BMP receptors. In vivo studies in C57BL/6J mice and of transplanted GFP-labeled Sca-1+CD45−CD11b− MSCs into bone marrow cavity of Rag2−/− immunodeficient mice showed PTH-enhanced phosphorylation of Smad1 and increased commitment of MSCs to osteoblast lineage, respectively. These data demonstrate that PTH-enhancement of MSCs differentiation to the osteoblast lineage occurs through a PTH and LRP6 dependent pathway by endocytosis of LRP6/PTH1R complex, allowing enhancement of BMP signaling.

Jab1 (Jun activation domain binding protein 1), integrated into COP9 signalosome complex (CSN), induces protein instability of many tumor suppressors and cell cycle regulators and is therefore a novel target in cancer therapy. Curcumin, an inhibitor of Jab1/CSN-associated kinase(s), has been reported to suppress tumor growth; however, curcumin is highly hydrophobic, and this feature prevents its usage as an antitumor drug. To increase the solubility and targeted delivery, we generated a water-soluble polyethylene glycol (PEG)-conjugated curcumin system, in which curcumin is covalently linked to PEG 35kD . PEGylated curcumin showed much greater reduction of cell growth than free curcumin in pancreatic cancer cells. Cells treated with PEGylated curcumin had increased arrest at the mitotic phase with the formation of abnormal multinucleated cells, indicating that this compound affects cell cycle progression, which may contribute to cell growth inhibition. The stabilities of Jab1 target proteins were also examined. PEGylated curcumin increased protein stability of these proteins in pancreatic cancer cells and directly inhibited the activity of Jab1/ CSN-associated kinases. Moreover, the inhibitory effect of PEGylated curcumin on cell proliferation was blunted in pancreatic cancer cells with Jab1 knockdown. The results suggest that PEGylated curcumin inhibits cell proliferation through suppression of Jab1/CSN activity. More importantly, the new compound sensitized pancreatic cancer cells to gemcitabine-induced apoptosis and cell proliferation inhibitory effects. Collectively, the PEGylated curcumin conjugate has much more potent effects on pancreatic cancer cell growth inhibition than free curcumin. The current study provides a biologic rationale to treat patients with pancreatic adenocarcinoma with the nontoxic phytochemical conjugated to PEG for systemic delivery.Pancreatic ductal adenocarcinoma represents greater than 80% of all pancreatic neoplasms with a death/incidence ratio of approximately 0.99 (Farrow and Evers, 2004;Brand and Mahr, 2005). Although gemcitabine currently is the most commonly used drug for treatment of pancreatic cancer (Burris et al

Most messenger RNA precursors (pre-mRNA) undergo cissplicing in which introns are excised and the adjoining exons from a single pre-mRNA are ligated together to form mature messenger RNA. This reaction is driven by a complex known as the spliceosome. Spliceosomes can also combine sequences from two independently transcribed pre-mRNAs in a process known as trans-splicing. Spliceosome-mediated RNA trans-splicing (SMaRT) is an emerging technology in which RNA pre-therapeutic molecules (PTMs) are designed to recode a specific pre-mRNA by suppressing cis-splicing while enhancing trans-splicing between the PTM and its premRNA target. This study examined the feasibility of SMaRT as a potential therapy for genetic diseases to correct mutations using cystic fibrosis (CF) as an example. We used several versions of a cystic fibrosis transmembrane conduc-