A new cell culture procedure has been developed that produces confluent primary hfRPE cultures with morphological and physiological characteristics of the native tissue. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied native human fetal and bovine RPE-choroid explants.

Rit is one of the original members of a novel Ras GTPase subfamily that uses distinct effector pathways to transform NIH 3T3 cells and induce pheochromocytoma cell (PC6) differentiation. In this study, we find that stimulation of PC6 cells by growth factors, including nerve growth factor (NGF), results in rapid and prolonged Rit activation. Ectopic expression of active Rit promotes PC6 neurite outgrowth that is morphologically distinct from that promoted by oncogenic Ras (evidenced by increased neurite branching) and stimulates activation of both the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase signaling pathways. Furthermore, Rit-induced differentiation is dependent upon both MAP kinase cascades, since MEK inhibition blocked Rit-induced neurite outgrowth, while p38 blockade inhibited neurite elongation and branching but not neurite initiation. Surprisingly, while Rit was unable to stimulate ERK activity in NIH 3T3 cells, it potently activated ERK in PC6 cells. This cell type specificity is explained by the finding that Rit was unable to activate C-Raf, while it bound and stimulated the neuronal Raf isoform, B-Raf. Importantly, selective down-regulation of Rit gene expression in PC6 cells significantly altered NGF-dependent MAP kinase cascade responses, inhibiting both p38 and ERK kinase activation. Moreover, the ability of NGF to promote neuronal differentiation was attenuated by Rit knockdown. Thus, Rit is implicated in a novel pathway of neuronal development and regeneration by coupling specific trophic factor signals to sustained activation of the B-Raf/ERK and p38 MAP kinase cascades.

Analyses of inflammation inducing Th cells revealed that a portion of these cells express both IFN‐γ and IL‐17, suggesting that Th1 and/or Th17 may switch their phenotype. Here, we examined phenotype retention in a system in which polarized Th1 or Th17 cells specific against hen egg lysozyme (HEL) induce ocular inflammation in recipient mice expressing HEL in their eyes. Whereas no switching to Th17 was detected in Th1 recipient eyes, substantial proportions of transferred Th17 expressed IFN‐γ, or both IFN‐γ and IL‐17 in Th17 recipient eyes. The phenotype switch of Th17 cells increased with time and was attributed to exposure to IL‐12 that is expressed in the inflamed eyes. Incubation in vitro with Th1 polarizing cocktail (IL‐2+IL‐12) converted most Th17 into IFN‐γ or IFN‐γ/IL‐17 expressing cells, but no reciprocal conversion was noted with Th1 cells. Furthermore, whereas Th1 cells did not express ROR‐γt following incubation with Th17 polarizing cocktail, Th17 expressed T‐bet following incubation with Th1 cocktail. Thus, polarized Th1 cells fully retain their phenotype, while Th17 switch and express IFN‐γ or both IFN‐γ and IL‐17, as well as T‐bet, following exposure to IL‐12. This study is supported by NEI/NIH intramural program.

Pituitary adenylate cyclase-activating polypeptide (PACAP38) stimulation results in the activation of G s ␣ protein-coupled receptors to regulate neuronal differentiation in a cyclic AMP (cAMP)-dependent manner. These pathways involve protein kinase A (PKA)-dependent processes, but a growing body of evidence indicates that cAMP also regulates cellular functions through PKA-independent signaling cascades. Here we show that the Rit small GTPase is regulated by PACAP38 in a cAMP-dependent but PKA-independent fashion. Rit activation results from stimulation of the cAMP-activated guanine nucleotide exchange factor Epac but does not appear to rely upon the activation of Rap GTPases, the accepted cellular Epac substrates. Although RNA interference studies demonstrated that Epac is required for PACAP38-mediated Rit activation, neither Epac1 nor Epac2 activates Rit directly, indicating that Epac signals to Rit through a novel mechanism in which Rap signaling is not essential. Loss-of-function analysis demonstrated that Rit makes an important contribution to PACAP38-mediated neuronal differentiation. Surprisingly, although Rit is required for sustained extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase signaling following nerve growth factor stimulation of pheochromocytoma 6 (PC6) cells, Rit silencing selectively suppressed PACAP38-elicited activation of p38, without obvious effects on ERK signaling in the same cells. Moreover, the ability of PACAP38 to stimulate CREB-dependent transcription and to promote neurite outgrowth was inhibited by Rit knockdown. Together, these studies identify an unsuspected connection between cAMP and Rit signaling pathways and imply that Rit can function downstream of G s ␣/cAMP/Epac in a novel signal transduction pathway necessary for PACAP38-mediated neuronal differentiation and CREB signaling.

Conserved structural motifs on pathogens trigger pattern recognition receptors present on APCs such as dendritic cells (DCs). An important class of such receptors is the Toll-like receptors (TLRs). TLR signaling triggers a cascade of events in DCs that includes modified chemokine and cytokine production, altered chemokine receptor expression, and changes in signaling through G protein-coupled receptors (GPCRs). One mechanism by which TLR signaling could modify GPCR signaling is by altering the expression of regulator of G protein signaling (RGS) proteins. In this study, we show that human monocyte-derived DCs constitutively express significant amounts of RGS2, RGS10, RGS14, RGS18, and RGS19, and much lower levels of RGS3 and RGS13. Engagement of TLR3 or TLR4 on monocyte-derived DCs induces RGS16 and RGS20, markedly increases RGS1 expression, and potently down-regulates RGS18 and RGS14 without modifying other RGS proteins. A similar pattern of Rgs protein expression occurred in immature bone marrow-derived mouse DCs stimulated to mature via TLR4 signaling. The changes in RGS18 and RGS1 expression are likely important for DC function, because both proteins inhibit G alpha(i)- and G alpha(q)-mediated signaling and can reduce CXC chemokine ligand (CXCL)12-, CC chemokine ligand (CCL)19-, or CCL21-induced cell migration. Providing additional evidence, bone marrow-derived DCs from Rgs1(-/-) mice have a heightened migratory response to both CXCL12 and CCL19 when compared with similar DCs prepared from wild-type mice. These results indicate that the level and functional status of RGS proteins in DCs significantly impact their response to GPCR ligands such as chemokines.