Regulatory role of ß-arrestin-2 in cholesterol processing in cystic fibrosis epithelial cells.

Cystic fibrosis (CF) cells exhibit an increase in the protein expression of ß-arrestin-2 (ßarr2) coincident with perinuclear accumulation of free cholesterol. Arrestins are proteins that both serve as broad signaling regulators and contribute to G-protein coupled receptor internalization after agonist stimulation. The hypothesis of this study is that ßarr2 is an important component in the mechanisms leading to cholesterol accumulation characteristic of CF cells. To test this hypothesis, epithelial cells stably expressing GFP-tagged ßarr2 (ßarr2-GFP) and respective GFP-expressing control cells (cont-GFP) were analyzed by filipin staining. The ßarr2-GFP cells show a late endosomal/lysosomal cholesterol accumulation that is identical to that seen in CF cells. This ßarr2-mediated accumulation is sensitive to Rp-cAMPS treatment, and depleting ßarr2 expression in CF-model cells by shRNA alleviates cholesterol accumulation compared with controls. Cftr/ßarr2 double knockout mice also exhibit wild-type (WT) levels of cholesterol synthesis, and WT profiles of signaling protein expression have previously been shown to be altered in CF due to cholesterol-related pathways. These data indicate a significant regulatory role for ßarr2 in the development of CF-like cholesterol accumulation and give further insight into cholesterol processing mechanisms. An impact of ßarr2 expression on Niemann-Pick type C-1 (NPC1)-containing organelle movement is proposed as the mechanism of ßarr2-mediated alterations on cholesterol processing. It is concluded that ßarr2 expression contributes to altered cholesterol trafficking observed in CF cells.

MAP kinase phosphatase 1 controls innate immune responses and suppresses endotoxic shock.

Septic shock is a leading cause of morbidity and mortality. However, genetic factors predisposing to septic shock are not fully understood. Excessive production of proinflammatory cytokines, particularly tumor necrosis factor (TNF)-alpha, and the resultant severe hypotension play a central role in the pathophysiological process. Mitogen-activated protein (MAP) kinase cascades are crucial in the biosynthesis of proinflammatory cytokines. MAP kinase phosphatase (MKP)-1 is an archetypal member of the dual specificity protein phosphatase family that dephosphorylates MAP kinase. Thus, we hypothesize that knockout of the Mkp-1 gene results in prolonged MAP kinase activation, augmented cytokine production, and increased susceptibility to endotoxic shock. Here, we show that knockout of Mkp-1 substantially sensitizes mice to endotoxic shock induced by lipopolysaccharide (LPS) challenge. We demonstrate that upon LPS challenge, Mkp-1-/- cells exhibit prolonged p38 and c-Jun NH2-terminal kinase activation as well as enhanced TNF-alpha and interleukin (IL)-6 production compared with wild-type cells. After LPS challenge, Mkp-1 knockout mice produce dramatically more TNF-alpha, IL-6, and IL-10 than do wild-type mice. Consequently, Mkp-1 knockout mice develop severe hypotension and multiple organ failure, and exhibit a remarkable increase in mortality. Our studies demonstrate that MKP-1 is a pivotal feedback control regulator of the innate immune responses and plays a critical role in suppressing endotoxin shock.

ß-arrestin-2 regulation of the cAMP response element binding protein.

Previous work demonstrated that cystic fibrosis (CF) cells exhibit an increase in cAMP-mediated signaling as a characteristic response to lost CFTR function. Evidence for increased cAMP-mediated signaling in CF included increased phosphorylation of the cAMP response element binding protein (CREB) and elevated ß-arrestin-2 (ßarr2) expression. However, subsequent studies reveal that CREB activation in CF cells is independent of protein kinase-A (PKA). The goal of this study is to test the hypothesis that elevated ßarr2 expression leads to increased CREB activation in a PKA-independent mechanism. ßarr2-GFP expressing tracheal epithelial cells (ßarr2-GFP) exhibit an increase of pCREB content and subsequent CRE activation compared to GFP expressing control cells. ßarr2 activation of the ERK cascade represents a candidate mechanism leading to CREB activation. ERK exhibits increased activation in ßarr2-GFP cells compared to cont-GFP cells, and ERK inhibition diminishes CRE activation in both GFP and ßarr2-GFP cells. To test directly whether CREB regulation in CF is ßarr2-dependent, nasal epithelium excised from wt mice (Cftr +/+; ßarr2 +/+), CF mice (Cftr -/-; ßarr2 +/+), and DKO mice (Cftr -/-; ßarr2 -/-) were analyzed for pCREB protein content. Removal of ßarr2 expression from CF mice reduces both pCREB and pERK content to wt levels. These data indicate that CF-related CREB regulation is mediated directly through ßarr2 expression via the ERK pathway.

Increased plasma membrane cholesterol in cystic fibrosis cells correlates with CFTR genotype and depends on de novo cholesterol synthesis.

BACKGROUND: Previous observations demonstrate that Cftr-null cells and tissues exhibit alterations in cholesterol processing including perinuclear cholesterol accumulation, increased de novo synthesis, and an increase in plasma membrane cholesterol accessibility compared to wild type controls. The hypothesis of this study is that membrane cholesterol accessibility correlates with CFTR genotype and is in part influenced by de novo cholesterol synthesis. METHODS: Electrochemical detection of cholesterol at the plasma membrane is achieved with capillary microelectrodes with a modified platinum coil that accepts covalent attachment of cholesterol oxidase. Modified electrodes absent cholesterol oxidase serves as a baseline control. Cholesterol synthesis is determined by deuterium incorporation into lipids over time. Incorporation into cholesterol specifically is determined by mass spectrometry analysis. All mice used in the study are on a C57Bl/6 background and are between 6 and 8 weeks of age. RESULTS: Membrane cholesterol measurements are elevated in both R117H and DeltaF508 mouse nasal epithelium compared to age-matched sibling wt controls demonstrating a genotype correlation to membrane cholesterol detection. Expression of wt CFTR in CF epithelial cells reverts membrane cholesterol to WT levels further demonstrating the impact of CFTR on these processes. In wt epithelial cell, the addition of the CFTR inhibitors, Gly H101 or CFTRinh-172, for 24 h surprisingly results in an initial drop in membrane cholesterol measurement followed by a rebound at 72 h suggesting a feedback mechanism may be driving the increase in membrane cholesterol. De novo cholesterol synthesis contributes to membrane cholesterol accessibility. CONCLUSIONS: The data in this study suggest that CFTR influences cholesterol trafficking to the plasma membrane, which when depleted, leads to an increase in de novo cholesterol synthesis to restore membrane content.

cAMP-mediated regulation of cholesterol accumulation in cystic fibrosis and Niemann-Pick type C cells.

The goal of this study was to identify a mechanism regulating cholesterol accumulation in cystic fibrosis (CF) cells. Both CFTR activation and expression are regulated by the cAMP pathway, and it is hypothesized that a feedback response involving this pathway may be involved in the phenotype of cholesterol accumulation. To examine the role of the cAMP pathway in cholesterol accumulation, we treated two CF model cell lines with the Rp diastereomer of adenosine 3',5'-cyclic monophosphorothioate (Rp-cAMPS) and visualized by filipin staining. Rp-cAMPS treatment eliminated cholesterol accumulation in CF cells, whereas 8-bromo-cAMP treatment led to cholesterol accumulation in wild-type cells. To confirm these findings in an independent model system, we also examined the role of cAMP in modulating cholesterol accumulation in Niemann-Pick type C (NPC) fibroblasts. Expression of the protein related to NPC, NPC1, is also directly regulated by cAMP; therefore, it is postulated that NPC cells exhibit the same cAMP-mediated control of cholesterol accumulation. Cholesterol accumulation in NPC cells also was reduced by the presence of Rp-cAMPS. Expression of beta-arrestin-2 (betaarr2), a marker of cellular response to cAMP signaling, was significantly elevated in CF model cells, Cftr(-/-) MNE, primary tissue obtained by nasal scrapes from CF subjects, and in NPC fibroblasts compared with respective controls.

Production of active recombinant mitogen-activated protein kinases through transient transfection of 293T cells.

Mitogen-activated protein (MAP) kinases are a family of serine/threonine protein kinases that play an important role in a myriad of cellular processes, including cell proliferation, differentiation, and apoptosis. Abnormal activation of MAP kinases has been shown to participate in a variety of human diseases which include cancer, septic shock, rheumatoid arthritis, diabetes, and cardiovascular diseases. Active MAP kinase enzymes are not only valuable for basic biomedical research but are also critical for the development of pharmacological inhibitors as therapeutic drugs in the treatment of relevant human diseases. MAP kinases produced in a bacterial system are poorly active due to a lack of proper phosphorylation at their characteristic threonine and tyrosine residues. To overcome these limitations, we have developed a mammalian expression system for high level expression and one-step purification of enzymatically MAP kinases. We cloned JNK1, p38, and p38-regulated MAP kinase-activated protein kinase-2 into the mammalian expression vector pEBG, and expressed these protein kinases as glutathione S-transferase fusion proteins in human embryonic kidney 293T cells through transient transfection. The protein kinases were activated in vivo through treating the transfected cells with sodium arsenite and affinity-purified using glutathione-Sepharose beads. The enzymatic activities of these protein kinases were demonstrated by Western blot analysis and in vitro kinase assays. Our results indicate that this system is an extremely powerful tool for generating valuable reagents, and could be very valuable for proteomic studies.

The role of mitogen-activated protein kinase phosphatase-1 in the response of alveolar macrophages to lipopolysaccharide: attenuation of proinflammatory cytokine biosynthesis via feedback control of p38.

Mitogen-activated protein (MAP) kinases are critical mediators of innate immune responses. In response to lipopolysaccharide (LPS), MAP kinases are rapidly activated and play an important role in the production of proinflammatory cytokines. Although a number of MAP kinase phosphatases (MKPs) have been identified, their roles in the control of cytokine production have not been well defined. In the present report, we investigated the role of MKP-1 in alveolar macrophages stimulated with LPS. We found that LPS triggered transient activation of three MAP kinase subfamilies, ERK, JNK, and p38, in both immortalized and primary murine alveolar macrophages. MKP-1 was rapidly induced by LPS, and its induction correlated with the dephosphorylation of these MAP kinases. Blocking MKP-1 with triptolide prolonged the activities of both JNK and p38 in immortalized alveolar macrophages. Stimulation of primary alveolar macrophages isolated from MKP-1-deficient mice with LPS resulted in a prolonged p38 phosphorylation compared with wild type alveolar macrophages. Accordingly, these MKP-1-deficient alveolar macrophages also mounted a more robust and rapid tumor necrosis factor alpha production than their wild type counterparts. Adenovirus-mediated MKP-1 overexpression significantly attenuated tumor necrosis factor alpha production in immortalized alveolar macrophages. Finally, MKP-1 was induced by a group of corticosteroids frequently prescribed for the treatment of inflammatory lung diseases, and the anti-inflammatory potencies of these drugs closely correlated with their abilities to induce MKP-1. Our studies indicated that MKP-1 plays an important role in dampening the inflammatory responses of alveolar macrophages. We speculate that MKP-1 may represent a novel target for therapeutic intervention of inflammatory lung diseases.