The IGF-I (insulin-like growth factor-I) signalling pathway responsible for regulation of proteoglycan synthesis in chondrocytes has not been defined and is the focus of the present study. Chondrocytes isolated from normal human articular cartilage were stimulated with IGF-I in monolayer culture or in suspension in alginate. IGF-I activated members of both the PI3K (phosphoinositide 3-kinase) pathway and the ERK (extracellular-signal-regulated kinase)/MAPK (mitogen-activated protein kinase) pathway. The PI3K inhibitors LY294002 and wortmannin blocked IGF-I-stimulated Akt phosphorylation without blocking ERK phosphorylation and this was associated with complete inhibition of proteoglycan synthesis. A decrease in IGF-I-stimulated proteoglycan synthesis was also observed upon inhibition of mTOR (mammalian target of rapamycin) and p70S6 kinase, both of which are downstream of Akt. The MEK (MAPK/ERK kinase) inhibitors PD98059 and U0126 blocked IGF-I-stimulated ERK phosphorylation but did not block the phosphorylation of Akt and did not decrease proteoglycan synthesis. Instead, in alginate- cultured chondrocytes, the MEK inhibitors increased IGF-I-stimulated proteoglycan synthesis when compared with cells treated with IGF-I alone. This is the first study to demonstrate that IGF-I stimulation of the PI3K signalling pathway is responsible for the ability of IGF-I to increase proteoglycan synthesis. Although IGF-I also activates the ERK/MAPK pathway, ERK activity is not required for proteoglycan synthesis and may serve as a negative regulator

Objective. The chondrocyte response to insulinlike growth factor 1 (IGF-1) is reduced with aging and in osteoarthritis (OA). IGF-1 signals through the phosphatidylinositol 3-kinase/Akt pathway. TRB3, a tribbles homolog, has been shown to inhibit IGF-1-mediated activation of Akt in HEK 293 cells. This study was undertaken to determine if TRB3 is expressed in chondrocytes, and whether the chondrocyte response to IGF-1 is reduced by TRB3.Methods. Human articular cartilage was obtained from normal tissue donors and from patients with OA at the time of knee replacement surgery. TRB3 was assessed in the tissue samples by reverse transcriptionpolymerase chain reaction, immunoblotting, and immunohistochemistry. Overexpression of TRB3 was induced by transient transfection to determine the effects of TRB3 on cell survival and proteoglycan synthesis.Results. TRB3 messenger RNA was detected in normal human chondrocytes. TRB3 protein levels were low in cells from normal cartilage but significantly increased in cells from OA cartilage. Incubation with 2 agents that induce endoplasmic reticulum stress, tunicamycin and thapsigargin, increased TRB3 levels in normal cells. Overexpression of TRB3 inhibited Akt phosphorylation and reduced chondrocyte survival and proteoglycan synthesis.Conclusion. These results are the first to demonstrate that TRB3 is present in human chondrocytes, and that the level of TRB3 is increased in OA cartilage and in isolated OA chondrocytes. Because it is an inhibitor of Akt activation, elevated TRB3 production could play a role in the increased cell death and reduced response to IGF-1 observed in OA cartilage.Aging does not directly cause osteoarthritis (OA); however, it is the most important risk factor in the development of the disease (1). A major contributing factor in the development of OA is a loss of the anabolic and catabolic homeostasis maintained by chondrocytes, leading to a loss of articular cartilage. The balance of anabolic and catabolic processes in cartilage depends on the local activity of regulatory factors such as cytokines and growth factors (2). Insulin-like growth factor 1 (IGF-1) has the ability to stimulate matrix synthesis (3-5), promote chondrocyte survival (6), and inhibit specific catabolic pathways (7,8). There is evidence that chondrocytes have a decreased response to IGF-1 with aging and also in OA (3,9-12), but the mechanisms involved are incompletely understood.IGF-1 stimulates the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathway as well as the Ras/ Raf/MEK/ERK pathway, by acting through the IGF-1 receptor (13). The activation of the IGF-1 receptor results in the activation of Shc and members of the insulin receptor substrate (IRS) family. After the phosphorylation of IRS and Shc, both the PI 3-kinase cascade and the ERK cascade are activated. The activation of PI 3-kinase leads to the activation of Akt (also called protein kinase B [PKB]), a serine/threonine kinase involved in Supported by NIH grant AG-16697.

In the last 10 years, there has been a relative explosion of new rodent systems that recapitulate both genetic and cellular lesions that lead to the development of pancreatic cancer. These models now need to be considered when selecting an appropriate in vivo system to study disease etiology, cell signaling, and drug development. The majority of these evaluations have used transplantation of cancer cells and the use of carcinogens, which still maintain their value when investigating human cancer and epigenetic contributors. Xenograft models utilize cultured or primary pancreatic cancer cells that are placed under the skin or implanted within the pancreas of immunocompromised mice. Carcinogen-induced systems rely on administration of certain chemicals to generate cellular changes that rapidly lead to pancreatic cancer. Genetically modified mice are more advanced in their design in that relevant genetic mutations can be inserted into mouse genomic DNA in both a conditional and inducible manner. Generation of mice that develop spontaneous pancreatic cancer from a targeted genetic mutation is a valuable research tool, considering the broad spectrum of genes and cell targets that can be used, producing a variety of neoplastic lesions and cancer that can reflect many aspects of human pancreatic ductal adenocarcinoma.

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