Adenosine A 2A Receptor Occupancy Stimulates Collagen Expression by Hepatic Stellate Cells via Pathways Involving Protein Kinase A, Src, and Extracellular Signal-Regulated Kinases 1/2 Signaling Cascade or p38 Mitogen-Activated Protein Kinase Signaling Pat

ABSTRACT Prior studies indicate that adenosine and the adenosine A 2A receptor play a role in hepatic fibrosis by a mechanism that has been proposed to involve direct stimulation of hepatic stellate cells (HSCs). The objective of this study was to determine whether primary hepatic stellate cells produce collagen in response to adenosine (via activation of adenosine A 2A receptors) and to further determine the signaling mechanisms involved in adenosine A 2A receptor-mediated promotion of collagen production. Cultured primary HSCs increase their collagen production after stimulation of the adenosine A 2A receptor in a dose-dependent fashion. Likewise, LX-2 cells, a human HSC line, increases expression of procollagen ␣I and procollagen ␣III mRNA and their translational proteins, collagen type I and type III, in response to pharmacological stimulation of adenosine A 2A receptors. Based on the use of pharmacological inhibitors of signal transduction, adenosine A 2A receptor-mediated stimulation of procollagen ␣I mRNA and collagen type I collagen expression were regulated by signal transduction involving protein kinase A, src, and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (erk), but surprisingly, adenosine A 2A receptor-mediated stimulation of procollagen ␣III mRNA and collagen type III protein expression depend on the activation of p38 mitogen-activated protein kinase (MAPK), findings confirmed by small interfering RNA-mediated knockdown of src, erk1, erk2, and p38 MAPK. These results indicate that adenosine A 2A receptors signal for increased collagen production by multiple signaling pathways. These results provide strong evidence in support of the hypothesis that adenosine receptors promote hepatic fibrosis, at least in part, via direct stimulation of collagen expression and that signaling for collagen production proceeds via multiple pathways

Dual Reporter Gene Imaging

The human and rodent sodium iodide symporters ( NIS ) have recently been cloned and are being investigated as potential therapeutic and reporter genes. We have extended this effort by constructing an internal ribosomal entry site (IRES)-linked human NIS (hNIS) -enhanced green fluorescent protein ( eGFP ) hybrid reporter gene for both nuclear and optical imaging. A self-inactivating retroviral vector, termed pQCNIG, containing hNIS-IRES-eGFP dual reporter gene, driven by a constitutive CMV promoter, was constructed and used to generate RG2-pQCNIG cells and RG2-pQCNIG tumors. 131 I-iodide and 99m TcO 4 -pertechnetate accumulation studies plus fluorescence microscopy and intensity assays were performed in vitro, and gamma camera imaging studies in RG2-pQCNIG and RG2 tumor-bearing athymic rats were performed. RG2-pQCNIG cells expressed high levels of hNIS protein and showed high intensity of eGFP fluorescence compared with RG2 wild-type cells. RG2-pQCNIG cells accumulated Na 131 I and 99m TcO 4 – to a 50:1 and a 170:1 tissue/medium ratio at 10 min, compared with 0.8:1.2 tissue/medium ratio in wild-type RG2 cells. A significant correlation between radiotracer accumulation and eGFP fluorescence intensity was demonstrated. RG2-pQCNIG and RG2 tumors were readily differentiated by in vivo gamma camera imaging; radiotracer uptake increased in RG2-pQCNIG but declined in RG2 tumors over the 50-min imaging period. Stomach and thyroid were the major organs of radionuclide accumulation. The IRES-linked hNIS-eGFP dual reporter gene is functional and stable in transduced RG2-pQCNIG cells. Optical and nuclear imaging of tumors produced from these cell lines provides the opportunity to monitor tumor growth and response to therapy. These studies indicate the potential for a wider application of hNIS reporter imaging and translation into patient studies using radioisotopes that are currently available for human use for both SPECT and PET imaging

HSP70-Inducible hNIS-IRES-eGFP Reporter Imaging: Response to Heat Shock

A retroviral vector pQHSP70/hNIS-IRES-eGFP (pQHNIG70) was constructed containing the hNIS-IRES-eGFP dual-reporter genes under the control of an inducible human heat shock protein (HSP)70 promoter and RG2-pQHSP70/hNIS-IRES-eGFP (RG2-pQHNIG70) transduced cells were generated. Heat-induced expression of both reporter genes in RG2-pQHNIG70 cells was validated by enhanced green fluorescent protein (eGFP) fluorescence-activated cell sorter, in vitro radiotracer assays, and immunoblot and immunocytochemistry. A 2.2- to 6.1-fold ( 131 I − ), a 6.1- to 14.4-fold ( 99m TcO 4 − ), and a 5.1- to 39-fold (fluorescence) increase above baseline was observed in response to graded hyperthermia (39–43°C). Increases in eGFP fluorescence and radiotracer uptake were first noted at 6 hours, reached a maximum at 24 hours, and fell toward baseline at 72 hours. A stable ratio of radiotracer uptake to eGFP fluorescence and to heat shock protein (HSP)70 protein was demonstrated over a wide range of expression levels, induced by different levels of heating. We also demonstrate that the local application of heat on RG2-pQHNIG70 xenografts can effectively induce hNIS and eGFP gene expression in vivo and that this expression can be efficiently visualized by fluorescence, scintigraphic, and micro–positron emission tomography imaging. Endogenous HSP70 protein and reporter expression was confirmed by postmortem tissue evaluations (immunoblot and immunohistochemistry). The pQHNIG70 reporter system can be used to study stress and drug responses in transduced cells and tissues