Non-Raft AC2 Defines a cAMP Signaling Compartment That Selectively Regulates IL-6 Expression in Airway Smooth Muscle Cells

Adenylyl cyclase (AC) isoforms differ in their tissue distribution, cellular localization, regulation, and protein interactions. Most cell types express multiple AC isoforms. We hypothesized that cAMP produced by different AC isoforms regulates unique cellular responses in human bronchial smooth muscle cells (BSMC). Overexpression of AC2, AC3, or AC6 had distinct effects on forskolin (Fsk)-induced expression of a number of known cAMP-responsive genes. These data show that different AC isoforms can differentially regulate gene expression. Most notable, overexpression and activation of AC2 enhanced interleukin 6 (IL-6) expression, but overexpression of AC3 or AC6 had no effect. IL-6 production by BSMC was induced by Fsk and select G protein-coupled receptor (GPCR) agonists, though IL-6 levels did not directly correlate with global cAMP levels. Treatment with PKA selective 6-Bnz-cAMP or Epac selective 8-CPT-2Me-cAMP cAMP analogs revealed a predominant role for PKA in cAMP-mediated induction of IL-6. IL-6 promoter mutations demonstrated that AP-1 and CRE transcription sites were required for Fsk to stimulate IL-6 expression. Our present study defines an AC2 cAMP signaling compartment that specifically regulates IL-6 expression in BSMC via Epac and PKA and demonstrates that other AC isoforms are excluded from this pool

[30] Induction of glucagon responsiveness in transformed MDCK cells unresponsive to glucagon

This chapter discusses a cloned line of Madin–Darby canine kidney (MDCK) cells, which were transformed with Harvey murine sarcoma virus. This line is maintained in continuous culture under the same conditions as the parental MDCK cells in Dulbecco's MEM, 5% fetal bovine serum, 5% CO2/95% air, with 80% humidity. The morphology of this transformed line is more fibroblastic than that of normal cells; in addition, a 21,000 Da protein (p21) coded by the virus has been identified on the inner surface of the plasma membrane of this transformed line. The growth characteristics of transformed MDCK cells are similar to normal cells; both grow equally well in serum free media. Like normal MDCK cells, the adenylate cyclase of the transformed line responds to a variety of hormones. However, transformation results in a selective loss of glucagon responsiveness because of the absence of glucagon binding sites at the cell surface. Thus, this cell line represents a good model system to examine factors that regulate the expression of differentiated functions.link_to_subscribed_fulltex

Revertants of Ha-MuSV-transformed MDCK cells express reduced levels of p21 and possess a more normal phenotype

Four subclones of the originally cloned Harvey murine sarcoma virus-transformed Madin Darby canine kidney (MDCK) cells have been isolated. These subclones fall into two general classes. Two subclones have a fibroblastic morphology, have lost the growth requirement for prostaglandin E 1 (PGE 1), do not respond to glucagon or vasopressin, and, in general, appear transformed. Two other subclones have epithelioid morphologies, are growth-stimulated by PGE 1, respond to vasopressin with an increase in intracellular cAMP. We propose that these cells represent revertants to a more non-transformed phenotype. Unlike normal cells, however, these revertants grow under anchorage-independent conditions, express detectable but reduceced amounts of the transforming gene product, p21, and grow in nude mice. The appearance of such revertants may be one cause of the observed heterogeneity of tumor cells.link_to_subscribed_fulltex

Characterization of hormone-sensitive Madin-Darby canine kidney cells

Cultured cells, especially the established lines, provide a continuous and homogeneous system for studying hormone action in intact cells. The cell line known as Madin–Darby canine kidney (MDCK) cells, derived from normal dog kidney more than 20 years ago, is ideal for this type of research, because it retains differentiated functions in culture and, in addition, responds to several hormones, including glucagon, vasopressin, β-adrenergic agonists, and prostaglandins. This chapter deals mainly with the optimal culture conditions for maintaining hormone responsiveness, the measurement of intracellular cyclic adenosine monophosphate (AMP). The chapter also discusses the characteristics of several types of hormone sensitivity in MDCK cells and concludes that MDCK cells have been used as a model system for studying hormone regulation of kidney functions. The availability of a differentiated, hormone-responsive cell line facilitates the studies of the cascade of biochemical events subsequent to cyclic AMP elevation and its correlation with specific kidney functions.link_to_subscribed_fulltex