Zhong Jian Shen

The peptidyl-prolyl isomerase Pin1 regulates the stability of granulocyte-macrophage colony-stimulating factor mRNA in activated eosinophils

The infiltration, accumulation and degranulation of eosinophils in the lung represents a hallmark of active asthma. In vivo or in vitro eosinophil activation triggers the secretion of the antiapoptotic cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF). We now identify Pin1, a cis-trans isomerase, as an essential component of the ribonucleoprotein complex responsible for GM-CSF mRNA stabilization, cytokine secretion and the survival of activated eosinophils. Pin1 regulated the association of the AU-rich element–binding proteins AUF1 and hnRNP C with GM-CSF mRNA, accelerating or slowing decay, respectively. These data indicate Pin1 is a key mediator of GM-CSF production.

Pin1 regulates TGF-β1 production by activated human and murine eosinophils and contributes to allergic lung fibrosis

Eosinophilic inflammation is a cornerstone of chronic asthma that often culminates in subepithelial fibrosis with variable airway obstruction. Pulmonary eosinophils (Eos) are a predominant source of TGF-beta1, which drives fibroblast proliferation and extracellular matrix deposition. We investigated the regulation of TGF-beta1 and show here that the peptidyl-prolyl isomerase (PPIase) Pin1 promoted the stability of TGF-beta1 mRNA in human Eos. In addition, Pin1 regulated cytokine production by both in vitro and in vivo activated human Eos. We found that Pin1 interacted with both PKC-alpha and protein phosphatase 2A, which together control Pin1 isomerase activity. Pharmacologic blockade of Pin1 in a rat asthma model selectively reduced eosinophilic pulmonary inflammation, TGF-beta1 and collagen expression, and airway remodeling. Furthermore, chronically challenged Pin1(-/-) mice showed reduced peribronchiolar collagen deposition compared with wild-type controls. These data suggest that pharmacologic suppression of Pin1 may be a novel therapeutic option to prevent airway fibrosis in individuals with chronic asthma.

Negative Regulation of Bone Morphogenetic Protein/Smad Signaling by Cas-interacting Zinc Finger Protein in Osteoblasts*

Bone morphogenetic protein (BMP) signaling regulates body axis determination, apoptosis, and differentiation of various types of cells including neuron, gut, and bone cells. However, the molecules involved in such BMP regulation of biological events have not been fully understood. Here, we examined the involvement of Cas-interacting zinc finger protein (CIZ) in the modulation of BMP2-induced osteoblastic cell differentiation. CIZ overexpression in osteoblastic MC3T3E1 cells suppressed BMP2-enhanced expression of alkaline phosphatase, osteocalcin, and type I collagen genes. Upstream analyses revealed that CIZ overexpression also suppressed BMP2-induced enhancement of the mRNA expression of Cbfa1, which is a critical transcription factor for osteoblastic differentiation. BMP-induced Smad1 and Smad5 activation of GCCG-mediated transcription was blocked in the presence of CIZ overexpression. CIZ overexpression alone in the absence of BMP2 moderately enhanced basal levels of Cbfa1 mRNA expression. CIZ overexpression also enhanced 1.8-kb Cbfa1 promoter activity in the absence of BMP2, whereas it suppressed the promoter activity in the presence of BMP2. Finally, CIZ overexpression suppressed the formation of mineralized nodules in osteoblastic cell cultures. These data indicate that CIZ is a novel type inhibitor of BMP/Smad signaling.

The peptidyl-prolyl isomerase Pin1 facilitates cytokine-induced survival of eosinophils by suppressing Bax activation

The mechanisms by which cytokine signals prevent the activation and mitochondrial targeting of the proapoptotic protein Bax are unclear. Here we show, using primary human eosinophils, that in the absence of the prosurvival cytokines granulocyte-macrophage colony-stimulating factor and interleukin 5, Bax spontaneously underwent activation and initiated mitochondrial disruption. Inhibition of Bax resulted in less eosinophil apoptosis, even in the absence of cytokines. Granulocyte-macrophage colony-stimulating factor induced activation of the kinase Erk1/2, which phosphorylated Thr167 of Bax; this facilitated new interaction of Bax with the prolyl isomerase Pin1. Blockade of Pin1 led to cleavage and mitochondrial translocation of Bax and caspase activation, regardless of the presence of cytokines. Our findings indicate that Pin1 is a key mediator of prosurvival signaling and is a regulator of Bax function.

The nucleocytoplasmic shuttling protein CIZ reduces adult bone mass by inhibiting bone morphogenetic protein–induced bone formation

Osteoporosis is a major health problem; however, the mechanisms regulating adult bone mass are poorly understood. Cas-interacting zinc finger protein (CIZ) is a nucleocytoplasmic shuttling protein that localizes at cell adhesion plaques that form where osteoblasts attach to substrate. To investigate the potential role of CIZ in regulating adult bone mass, we examined the bones in CIZ-deficient mice. Bone volume was increased and the rates of bone formation were increased in CIZ-deficient mice, whereas bone resorption was not altered. CIZ deficiency enhanced the levels of mRNA expression of genes encoding proteins related to osteoblastic phenotypes, such as alkaline phosphatase (ALP) as well as osterix mRNA expression in whole long bones. Bone marrow cells obtained from the femora of CIZ-deficient mice revealed higher ALP activity in culture and formed more mineralized nodules than wild-type cells. CIZ deficiency enhanced bone morphogenetic protein (BMP)–induced osteoblastic differentiation in bone marrow cells in cultures, indicating that BMP is the target of CIZ action. CIZ deficiency increased newly formed bone mass after femoral bone marrow ablation in vivo. Finally, BMP-2–induced bone formation on adult mouse calvariae in vivo was enhanced by CIZ deficiency. These results establish that CIZ suppresses the levels of adult bone mass through inhibition of BMP-induced activation of osteoblasts.

The Peptidyl-Prolyl Isomerase Pin1 Regulates Granulocyte-Macrophage Colony-Stimulating Factor mRNA Stability in T Lymphocytes

Cytokine production is associated with both the normal and pathologic inflammatory response to injury. Previous studies have shown that the immunosuppressants cyclosporin A or FK506, which interact with the peptidyl-propyl isomerases cyclophilin A and FK506-binding protein (FKBP12), respectively, block cytokine expression. A third member of the peptidyl-propyl isomerase family, Pin1 is expressed by immune and other cells. Pin1 has been implicated in cell cycle progression, is overexpressed in human tumors, and may rescue neurons from τ-associated degeneration. However, the role of Pin1 in the immune system remains largely unknown. In this study, we analyze the role of Pin1 in GM-CSF expression by human PBMC and CD4+ lymphocytes. We show that Pin1 isomerase activity is necessary for activation-dependent, GM-CSF mRNA stabilization, accumulation, and protein secretion, but not non-AU-rich elements containing cytokine mRNAs, including TGF-β and IL-4. Mechanistically, Pin1 mediated the association of the AU-rich element-binding protein, AUF1, with GM-CSF mRNA, which determined the rate of decay by the exosome.

FLASH Coordinates NF-κB Activity via TRAF2

FLASH is a protein recently shown to interact with the death effector domain of caspase-8 and is likely to be a component of the death-inducing signaling complex in receptor-mediated apoptosis. Here we show that antisense oligonucleotide-induced inhibition of FLASH expression abolished TNF-α-induced activation of NF-κB in HEK293 cells, as determined by luciferase reporter gene expression driven by a NF-κB responsive promoter. Conversely, overexpression of FLASH dose-dependently activated NF-κB, an effect suppressed by dominant negative mutants of TRAF2, NIK, and IKKα, and partially by those of TRAF5 and TRAF6. TRAF2 was co-immunoprecipitated with FLASH from the cell extracts of HEK293 cells or HeLa cells stably expressing exogenous FLASH (HeLa/HA-FLASH). Furthermore, serial deletion mapping demonstrated that a domain spanning the residues 856–1191 of FLASH activated NF-κB as efficiently as the full-length and could directly bind to TRAF2in vitro and in the transfected cells. Taken together, these results suggest that FLASH coordinates downstream NF-κB activity via a TRAF2-dependent pathway in the TNF-α signaling.

Pin1 Modulates the Type 1 Immune Response

Background/Abstract Immune responses initiated by T cell receptor (TCR) and costimulatory molecule mediated signaling culminate in maximal cytokine mRNA production and stability. The transcriptional responses to co-stimulatory T cell signalling involve calcineurin and NF-AT, which can be antagonized by interference with the cis-trans peptidyl-prolyl isomerases (PPIase), cyclophilin A and FKBP. Signalling molecules downstream of CD28 which are essential for the stabilization of cytokine mRNAs are largely unknown. Methodology/Principal Findings We now show that Pin1, a third member of the PPIase family mediates the post-transcriptional regulation of Th1 cytokines by activated T cells. Blockade of Pin1 by pharmacologic or genetic means greatly attenuated IFN-γ, IL-2 and CXCL-10 mRNA stability, accumulation and protein expression after cell activation. In vivo, Pin1 blockade prevented both the acute and chronic rejection of MHC mismatched, orthotopic rat lung transplants by reducing the expression of IFN-γ and CXCL-10. Combined transcriptional and post-transcriptional blockade with cyclosporine A and the Pin1 inhibitor, juglone, was synergistic. Conclusions/Significance These data suggest Pin1 inhibitors should be explored for use as immunosuppressants and employed with available calcineurin inhibitors to reduce toxicity and enhance effectiveness.

Pin1 Protein Regulates Smad Protein Signaling and Pulmonary Fibrosis

Background: Interstitial pulmonary fibrosis is caused by the excess production of extracellular matrix (ECM) by Fb in response to TGF-β1. Results: The peptidyl-prolyl isomerase Pin1 modulates the production of many pro- and antifibrogenic cytokines and ECM after bleomycin injury. Conclusion: Pin1 controls Smad6 function. Significance: Pin1 may be a therapeutic target to prevent pathologic lung scarring. Interstitial pulmonary fibrosis is caused by the excess production of extracellular matrix (ECM) by Fb in response to TGF-β1. Here, we show that the peptidyl-prolyl isomerase Pin1 modulates the production of many pro- and antifibrogenic cytokines and ECM. After acute, bleomycin injury, Pin1−/− mice showed reduced, pulmonary expression of collagens, tissue inhibitors of metalloproteinases, and fibrogenic cytokines but increased matrix metalloproteinases, compared with WT mice, despite similar levels of inflammation. In primary fibroblasts, Pin1 was required for TGF-β-induced phosphorylation, nuclear translocation, and transcriptional activity of Smad3. In Pin1−/− cells, inhibitory Smad6 was found in the cytoplasm rather than nucleus. Smad6 knockdown in Pin1−/− fibroblasts restored TGF-β-induced Smad3 activation, translocation, and target gene expression. Therefore, Pin1 is essential for normal Smad6 function and ECM production in response to injury or TGF-β and thus may be an attractive therapeutic target to prevent excess scarring in diverse lung diseases.

IL-3 Maintains Activation of the p90S6K/RPS6 Pathway and Increases Translation in Human Eosinophils

IL-5 is a major therapeutic target to reduce eosinophilia. However, all of the eosinophil-activating cytokines, such as IL-5, IL-3, and GM-CSF, are typically present in atopic diseases, including allergic asthma. As a result of the functional redundancy of these three cytokines on eosinophils and the loss of IL-5R on airway eosinophils, it is important to take IL-3 and GM-CSF into account to efficiently reduce tissue eosinophil functions. Moreover, these three cytokines signal through a common β-chain receptor but yet differentially affect protein production in eosinophils. Notably, the increased ability of IL-3 to induce the production of proteins, such as semaphorin-7A, without affecting mRNA levels suggests a unique influence of IL-3 on translation. The purpose of this study was to identify the mechanisms by which IL-3 distinctively affects eosinophil function compared with IL-5 and GM-CSF, with a focus on protein translation. Peripheral blood eosinophils were used to study intracellular signaling and protein translation in cells activated with IL-3, GM-CSF, or IL-5. We establish that, unlike GM-CSF or IL-5, IL-3 triggers prolonged signaling through activation of ribosomal protein S6 (RPS6) and the upstream kinase 90-kDa ribosomal S6 kinase (p90S6K). Blockade of p90S6K activation inhibited phosphorylation of RPS6 and IL-3–enhanced semaphorin-7A translation. Furthermore, in an allergen-challenged environment, in vivo phosphorylation of RPS6 and p90S6K was enhanced in human airway compared with circulating eosinophils. Our findings provide new insights into the mechanisms underlying differential activation of eosinophils by IL-3, GM-CSF, and IL-5. These observations identify IL-3 and its downstream intracellular signals as novel targets that should be considered to modulate eosinophil functions.