CLP36 promotes p53 deficient sarcoma progression through suppression of atrophin-1 interacting protein-4 (AIP-4)-dependent degradation of YAP1.

Background: p53 deficiency is a key causal factor for tumor development and progression. p53 acts in this process through, at least in part, cooperation with YAP1 but the underlying molecular mechanism is incompletely understood. In this paper, we show that CLP36, an actinin-binding cytoskeletal protein, links p53 deficiency to up-regulation of YAP1 expression and sarcoma progression. Methods: Immunohistochemical staining and Western blotting were used to investigate the effect of p53 deficiency on CLP36 expression in sarcoma tissues and cells. Furthermore, molecular, cellular, and genetic knockout and knockdown approaches were employed to investigate the functions of CLP36 in regulation of sarcoma cell behavior in culture and tumor growth in mice. Finally, biochemical approaches were used to investigate the molecular mechanism by which CLP36 regulates the malignant behavior of p53 deficient sarcoma cells. Results: We have found that the expression of CLP36 is up-regulated in response to loss of p53 in sarcoma tissues and cells. Depletion of CLP36 inhibited malignant behavior of p53 deficient sarcoma cells. Furthermore, knockout of CLP36 in mice markedly inhibited p53 deficiency-induced tumorigenesis and improved the survival of the p53 deficient mice. Mechanistically, CLP36 promoted p53 deficiency-induced tumorigenesis through inhibition of E3 ligase atrophin-1 interacting protein-4 (AIP-4)-dependent proteasomal degradation of YAP1 and consequently increase of YAP1 expression. Conclusions: Our results reveal a crucial role of CLP36 in linking p53 deficiency to up-regulation of YAP1 expression and sarcoma progression. Our findings suggest that therapeutic targeting the CLP36/YAP1 signaling axis may provide an effective strategy for alleviation of p53 deficient sarcoma progression.

A gain-of-function mutation in the ITPR1 gating domain causes male infertility in mice.

Inositol 1,4,5-trisphosphate receptor 1 (ITPR1) is an intracellular Ca2+ release channel critical for numerous cellular processes. Despite its ubiquitous physiological significance, ITPR1 mutations have thus far been linked to primarily movement disorders. Surprisingly, most disease-associated ITPR1 mutations generate a loss of function. This leaves our understanding of ITPR1-associated pathology oddly one-sided, as little is known about the pathological consequences of ITPR1 gain of function (GOF). To this end, we generated an ITPR1 gating domain mutation (D2594K) that substantially enhanced the inositol trisphosphate (IP3 )-sensitivity of ITPR1, and a mouse model expressing this ITPR1-D2594K+/- GOF mutation. We found that heterozygous ITPR1-D2594K+/- mutant mice exhibited male infertility, azoospermia, and acrosome loss. Furthermore, we functionally characterized a human ITPR1 variant V494I identified in the UK Biobank database as potentially associated with disorders of the testis. We found that the ITPR1-V494I variant significantly enhanced IP3 -induced Ca2+ release in HEK293 cells. Thus, ITPR1 hyperactivity may increase the risk of testicular dysfunction.

Circular RNA ciRs-126 promotes hypoxia/reoxygenation cardiac injury possibly through miR-21.

BACKGROUND: This study aimed to analyze the role of circular RNA ciRs-126 in hypoxia/reoxygenation cardiac injury (H/R). METHODS: Expression of ciRs-126 and miR-21 in plasma samples from patients with H/R and healthy controls was determined by RT-qPCR. Correlations were analyzed by linear regression. Overexpression of ciRs-126 and miR-21 was achieved in cardiomyocytes to explore their crosstalk. The roles of ciRs-126 and miR-21 in H/R-induced apoptosis of cardiomyocytes were analyzed using cell apoptosis assay. RESULTS: CiRs-126 was upregulated and miR-21 was downregulated in H/R patients. They were inversely correlated across plasma samples from H/R patients. In H/R cardiomyocytes, ciRs-126 was upregulated and miR-21 was downregulated. In cardiomyocytes, ciRs-126 overexpression decreased miR-21 level and reduced the inhibitory effects of miR-21 overexpression on H/R-induced cell apoptosis. CONCLUSIONS: Circular RNA ciRs-126 may suppress miR-21 expression to promote H/R cardiac injury.

O-GlcNAcylation Is Essential for Autophagy in Cardiomyocytes.

Since both O-GlcNAcylation and autophagy sense intracellular nutrient level, the alteration of those two pathways plays substantial roles in the progression of heart failure. Hence, determining the relationship between O-GlcNAcylation and autophagy is imperative to understand, prevent, and treat heart failure. However, the mechanism on how O-GlcNAcylation regulates autophagy in the heart is poorly investigated. In this study, we demonstrated that O-GlcNAcylation is required for autophagy in cardiomyocytes by utilizing an O-linked ß-N-acetylglucosamine transferase (OGT) cardiomyocyte-specific knockout mouse model for the first time. We also identified that OGT might regulate the initiation of autophagy in cardiomyocytes through promoting the activity of ULK1 by O-GlcNAcylation. In conclusion, our findings provide new insights into the molecular mechanisms underlying heart dysfunction and benefit the development of treatments for heart failure.

O-linked ß-N-acetylglucosamine transferase plays an essential role in heart development through regulating angiopoietin-1.

O-linked N-acetylglucosamine (GlcNAc) transferase (OGT) is the only enzyme catalyzing O-GlcNAcylation. Although it has been shown that OGT plays an essential role in maintaining postnatal heart function, its role in heart development remains unknown. Here we showed that loss of OGT in early fetal cardiomyocytes led to multiple heart developmental defects including hypertrabeculation, biventricular dilation, atrial septal defects, ventricular septal defects, and defects in coronary vessel development. In addition, RNA sequencing revealed that Angiopoietin-1, required within cardiomyocytes for both myocardial and coronary vessel development, was dramatically downregulated in cardiomyocyte-specific OGT knockout mouse hearts. In conclusion, our data demonstrated that OGT plays an essential role in regulating heart development through activating expression of cardiomyocyte Angiopoietin-1.

Apical-Basal Polarity Signaling Components, Lgl1 and aPKCs, Control Glutamatergic Synapse Number and Function.

Normal synapse formation is fundamental to brain function. We show here that an apical-basal polarity (A-BP) protein, Lgl1, is present in the postsynaptic density and negatively regulates glutamatergic synapse numbers by antagonizing the atypical protein kinase Cs (aPKCs). A planar cell polarity protein, Vangl2, which inhibits synapse formation, was decreased in synaptosome fractions of cultured cortical neurons from Lgl1 knockout embryos. Conditional knockout of Lgl1 in pyramidal neurons led to reduction of AMPA/NMDA ratio and impaired plasticity. Lgl1 is frequently deleted in Smith-Magenis syndrome (SMS). Lgl1 conditional knockout led to increased locomotion, impaired novel object recognition and social interaction. Lgl1+/- animals also showed increased synapse numbers, defects in open field and social interaction, as well as stereotyped repetitive behavior. Social interaction in Lgl1+/- could be rescued by NMDA antagonists. Our findings reveal a role of apical-basal polarity proteins in glutamatergic synapse development and function and also suggest a potential treatment for SMS patients with Lgl1 deletion.

P209L mutation in Bag3 does not cause cardiomyopathy in mice.

Bcl-2-associated athanogene 3 (BAG3) is a cochaperone protein and a central player of the cellular protein quality control system. BAG3 is prominently expressed in the heart and plays an essential role in cardiac protein homeostasis by interacting with chaperone heat shock proteins (HSPs) in large, functionally distinct multichaperone complexes. The BAG3 mutation of proline 209 to leucine (P209L), which resides in a critical region that mediates the direct interaction between BAG3 and small HSPs (sHSPs), is associated with cardiomyopathy in humans. However, the mechanism by which the BAG3 P209L missense mutation leads to cardiomyopathy remains unknown. To determine the molecular basis underlying the cardiomyopathy caused by the BAG3 P209L mutation, we generated a knockin (KI) mouse model in which the endogenous Bag3 gene was replaced with mutant Bag3 containing the P215L mutation, which is equivalent to the human P209L mutation. We performed physiological, histological, and biochemical analyses of Bag3 P209L KI mice to determine the functional, morphological, and molecular consequences of the P209L mutation. We found that Bag3 P209L KI mice exhibited normal cardiac function and morphology up to 16 mo of age. Western blot analysis further revealed that levels of sHSPs, stress-inducible HSPs, ubiquitinated proteins, and autophagy were unaffected in P209L mutant mouse hearts. In conclusion, the P209L mutation in Bag3 does not cause cardiomyopathy in mice up to 16 mo of age under baseline conditions. NEW & NOTEWORTHY Bcl-2-associated athanogene 3 (BAG3) P209L mutation is associated with human cardiomyopathy. A recent study reported that transgenic mice overexpressing human BAG3 P209L in cardiomyocytes have cardiac dysfunction. In contrast, our P209L mice that express mutant BAG3 at the same level as that of wild-type mice displayed no overt phenotype. Our results suggest that human cardiomyopathy may result from species-specific requirements for the conserved motif that is disrupted by P209L mutation or from genetic background-dependent effects.

aPKCζ-dependent Repression of Yap is Necessary for Functional Restoration of Irradiated Salivary Glands with IGF-1.

Xerostomia and salivary hypofunction often result as a consequence of radiation therapy for head and neck cancers, which are diagnosed in roughly 60,000 individuals every year in the U.S. Due to the lack of effective treatments for radiation-induced salivary hypofunction, stem cell-based therapies have been suggested to regenerate the irradiated salivary glands. Pharmacologically, restoration of salivary gland function has been accomplished in mice by administering IGF-1 shortly after radiation treatment, but it is not known if salivary stem and progenitor cells play a role. We show that radiation inactivates aPKCζ and promotes nuclear redistribution of Yap in a population of label-retaining cells in the acinar compartment of the parotid gland (PG)- which comprises a heterogeneous pool of salivary progenitors. Administration of IGF-1 post-radiation maintains activation of aPKCζ and partially rescues Yap's cellular localization in label retaining cells, while restoring salivary function. Finally, IGF-1 fails to restore saliva production in mice lacking aPKCζ, demonstrating the importance of the kinase as a potential therapeutic target.

HSPB7 is indispensable for heart development by modulating actin filament assembly.

Small heat shock protein HSPB7 is highly expressed in the heart. Several mutations within HSPB7 are associated with dilated cardiomyopathy and heart failure in human patients. However, the precise role of HSPB7 in the heart is still unclear. In this study, we generated global as well as cardiac-specific HSPB7 KO mouse models and found that loss of HSPB7 globally or specifically in cardiomyocytes resulted in embryonic lethality before embryonic day 12.5. Using biochemical and cell culture assays, we identified HSPB7 as an actin filament length regulator that repressed actin polymerization by binding to monomeric actin. Consistent with HSPB7's inhibitory effects on actin polymerization, HSPB7 KO mice had longer actin/thin filaments and developed abnormal actin filament bundles within sarcomeres that interconnected Z lines and were cross-linked by α-actinin. In addition, loss of HSPB7 resulted in up-regulation of Lmod2 expression and mislocalization of Tmod1. Furthermore, crossing HSPB7 null mice into an Lmod2 null background rescued the elongated thin filament phenotype of HSPB7 KOs, but double KO mice still exhibited formation of abnormal actin bundles and early embryonic lethality. These in vivo findings indicated that abnormal actin bundles, not elongated thin filament length, were the cause of embryonic lethality in HSPB7 KOs. Our findings showed an unsuspected and critical role for a specific small heat shock protein in directly modulating actin thin filament length in cardiac muscle by binding monomeric actin and limiting its availability for polymerization.

Loss-of-function mutations in co-chaperone BAG3 destabilize small HSPs and cause cardiomyopathy.

Defective protein quality control (PQC) systems are implicated in multiple diseases. Molecular chaperones and co-chaperones play a central role in functioning PQC. Constant mechanical and metabolic stress in cardiomyocytes places great demand on the PQC system. Mutation and downregulation of the co-chaperone protein BCL-2-associated athanogene 3 (BAG3) are associated with cardiac myopathy and heart failure, and a BAG3 E455K mutation leads to dilated cardiomyopathy (DCM). However, the role of BAG3 in the heart and the mechanisms by which the E455K mutation leads to DCM remain obscure. Here, we found that cardiac-specific Bag3-KO and E455K-knockin mice developed DCM. Comparable phenotypes in the 2 mutants demonstrated that the E455K mutation resulted in loss of function. Further experiments revealed that the E455K mutation disrupted the interaction between BAG3 and HSP70. In both mutants, decreased levels of small heat shock proteins (sHSPs) were observed, and a subset of proteins required for cardiomyocyte function was enriched in the insoluble fraction. Together, these observations suggest that interaction between BAG3 and HSP70 is essential for BAG3 to stabilize sHSPs and maintain cardiomyocyte protein homeostasis. Our results provide insight into heart failure caused by defects in BAG3 pathways and suggest that increasing BAG3 protein levels may be of therapeutic benefit in heart failure.

Postnatal Loss of Kindlin-2 Leads to Progressive Heart Failure.

BACKGROUND: The striated muscle costamere, a multiprotein complex at the boundary between the sarcomere and the sarcolemma, plays an integral role in maintaining striated muscle structure and function. Multiple costamere-associated proteins, such as integrins and integrin-interacting proteins, have been identified and shown to play an increasingly important role in the pathogenesis of human cardiomyopathy. Kindlin-2 is an adaptor protein that binds to the integrin ß cytoplasmic tail to promote integrin activation. Genetic deficiency of Kindlin-2 results in embryonic lethality, and knockdown of the Kindlin-2 homolog in Caenorhabditis elegans and Danio rerio suggests that it has an essential role in integrin function and normal muscle structure and function. The precise role of Kindlin-2 in the mammalian cardiac myocyte remains to be determined. METHODS AND RESULTS: The current studies were designed to investigate the role of Kindlin-2 in the mammalian heart. We generated a series of cardiac myocyte-specific Kindlin-2 knockout mice with excision of the Kindlin-2 gene in either developing or adult cardiac myocytes. We found that mice lacking Kindlin-2 in the early developing heart are embryonic lethal. We demonstrate that deletion of Kindlin-2 at late gestation or in adult cardiac myocytes resulted in heart failure and premature death, which were associated with enlargement of the heart and extensive fibrosis. In addition, integrin ß1D protein expression was significantly downregulated in the adult heart. CONCLUSIONS: Kindlin-2 is required to maintain integrin ß1D protein stability. Postnatal loss of Kindlin-2 from cardiac myocytes leads to progressive heart failure, showing the importance of costameric proteins like Kindlin-2 for homeostasis of normal heart function.

Cypher and Enigma homolog protein are essential for cardiac development and embryonic survival.

BACKGROUND: The striated muscle Z-line, a multiprotein complex at the boundary between sarcomeres, plays an integral role in maintaining striated muscle structure and function. Multiple Z-line-associated proteins have been identified and shown to play an increasingly important role in the pathogenesis of human cardiomyopathy. Cypher and its close homologue, Enigma homolog protein (ENH), are 2 Z-line proteins previously shown to be individually essential for maintenance of postnatal cardiac function and stability of the Z-line during muscle contraction, but dispensable for cardiac myofibrillogenesis and development. METHODS AND RESULTS: The current studies were designed to test whether Cypher and ENH play redundant roles during embryonic development. Here, we demonstrated that mice lacking both ENH and Cypher exhibited embryonic lethality and growth retardation. Lethality in double knockout embryos was associated with cardiac dilation and abnormal Z-line structure. In addition, when ENH was ablated in conjunction with selective ablation of either Cypher short isoforms (CypherS), or Cypher long isoforms (CypherL), only the latter resulted in embryonic lethality. CONCLUSIONS: Cypher and ENH redundantly play an essential role in sustaining Z-line structure from the earliest stages of cardiac function, and are redundantly required to maintain normal embryonic heart function and embryonic viability.

Normalization of Naxos plakoglobin levels restores cardiac function in mice.

Arrhythmogenic cardiomyopathy (AC) is associated with mutations in genes encoding intercalated disc proteins and ultimately results in sudden cardiac death. A subset of patients with AC have the autosomal recessive cardiocutaneous disorder Naxos disease, which is caused by a 2-base pair deletion in the plakoglobin-encoding gene JUP that results in a truncated protein with reduced expression. In mice, cardiomyocyte-specific plakoglobin deficiency recapitulates many aspects of human AC, and overexpression of the truncated Naxos-associated plakoglobin also results in an AC-like phenotype; therefore, it is unclear whether Naxos disease results from loss or gain of function consequent to the plakoglobin mutation. Here, we generated 2 knockin mouse models in which endogenous Jup was engineered to express the Naxos-associated form of plakoglobin. In one model, Naxos plakoglobin bypassed the nonsense-mediated mRNA decay pathway, resulting in normal levels of the truncated plakoglobin. Moreover, restoration of Naxos plakoglobin to WT levels resulted in normal heart function. Together, these data indicate that a gain of function in the truncated form of the protein does not underlie the clinical phenotype of patients with Naxos disease and instead suggest that insufficiency of the truncated Naxos plakoglobin accounts for disease manifestation. Moreover, these results suggest that increasing levels of truncated or WT plakoglobin has potential as a therapeutic approach to Naxos disease.

Generation and characterization of a mouse model harboring the exon-3 deletion in the cardiac ryanodine receptor.

A large genomic deletion in human cardiac ryanodine receptor (RYR2) gene has been detected in a number of unrelated families with various clinical phenotypes, including catecholaminergic polymorphic ventricular tachycardia (CPVT). This genomic deletion results in an in-frame deletion of exon-3 (Ex3-del). To understand the underlying disease mechanism of the RyR2 Ex3-del mutation, we generated a mouse model in which the RyR2 exon-3 sequence plus 15-bp intron sequences flanking exon-3 were deleted. Heterozygous Ex3-del mice (Ex3-del+/-) survived, but no homozygous Ex3-del mice were born. Unexpectedly, the Ex3-del+/- mice are not susceptible to CPVT. Ex3-del+/- cardiomyocytes exhibited similar amplitude but altered dynamics of depolarization-induced Ca2+ transients compared to wild type (WT) cells. Immunoblotting analysis revealed markedly reduced expression of RyR2 protein in the Ex3-del+/- mutant heart, indicating that Ex3-del has a major impact on RyR2 protein expression in mice. Cardiac specific, conditional knockout of the WT RyR2 allele in Ex3-del+/- mice led to bradycardia and death. Thus, the absence of CPVT and other phenotypes in Ex3-del+/- mice may be attributable to the predominant expression of the WT RyR2 allele as a result of the markedly reduced expression of the Ex3-del mutant allele. The effect of Ex3-del on RyR2 protein expression is discussed in relation to the phenotypic variability in individuals with the RyR2 exon-3 deletion.

Cypher/ZASP is a novel A-kinase anchoring protein.

PKA signaling is important for the post-translational modification of proteins, especially those in cardiomyocytes involved in cardiac excitation-contraction coupling. PKA activity is spatially and temporally regulated through compartmentalization by protein kinase A anchoring proteins. Cypher/ZASP, a member of PDZ-LIM domain protein family, is a cytoskeletal protein that forms multiprotein complexes at sarcomeric Z-lines. It has been demonstrated that Cypher/ZASP plays a pivotal structural role in the structural integrity of sarcomeres, and several of its mutations are associated with myopathies including dilated cardiomyopathy. Here we show that Cypher/ZASP, interacting specifically with the type II regulatory subunit RIIα of PKA, acted as a typical protein kinase A anchoring protein in cardiomyocytes. In addition, we show that Cypher/ZASP itself was phosphorylated at Ser(265) and Ser(296) by PKA. Furthermore, the PDZ domain of Cypher/ZASP interacted with the L-type calcium channel through its C-terminal PDZ binding motif. Expression of Cypher/ZASP facilitated PKA-mediated phosphorylation of the L-type calcium channel in vitro. Additionally, the phosphorylation of the L-type calcium channel at Ser(1928) induced by isoproterenol was impaired in neonatal Cypher/ZASP-null cardiomyocytes. Moreover, Cypher/ZASP interacted with the Ser/Thr phosphatase calcineurin, which is a phosphatase for the L-type calcium channel. Taken together, our data strongly suggest that Cypher/ZASP not only plays a structural role for the sarcomeric integrity, but is also an important sarcomeric signaling scaffold in regulating the phosphorylation of channels or contractile proteins.

No contribution of IP3-R(2) to disease phenotype in models of dilated cardiomyopathy or pressure overload hypertrophy.

BACKGROUND: We investigated the contribution of inositol(1,4,5)-trisphosphate (Ins(1,4,5)P3 [IP3]) receptors (IP3-R) to disease progression in mouse models of dilated cardiomyopathy (DCM) and pressure overload hypertrophy. Mice expressing mammalian sterile 20-like kinase and dominant-negative phosphatidylinositol-3-kinase in heart (Mst1×dn-PI3K-2Tg; DCM-2Tg) develop severe DCM and conduction block, associated with increased expression of type 2 IP3-R (IP3-R(2)) and heightened generation of Ins(1,4,5)P3. Similar increases in Ins(1,4,5)P3 and IP3-R(2) are caused by transverse aortic constriction. METHODS AND RESULTS: To evaluate the contribution of IP3-R(2) to disease progression, the DCM-2Tg mice were further crossed with mice in which the type 2 IP3-R (IP3-R(2)-/-) had been deleted (DCM-2Tg×IP3-R(2)-/-) and transverse aortic constriction was performed on IP3-R(2)-/- mice. Hearts from DCM-2Tg mice and DCM-2Tg×IP3-R(2)-/- were similar in terms of chamber dilatation, atrial enlargement, and ventricular wall thinning. Electrophysiological changes were also similar in the DCM-2Tg mice, with and without IP3-R(2). Deletion of IP3-R(2) did not alter the progression of heart failure, because DCM-2Tg mice with and without IP3-R(2) had similarly reduced contractility, increased lung congestion, and atrial thrombus, and both strains died between 10 and 12 weeks of age. Loss of IP3-R(2) did not alter the progression of hypertrophy after transverse aortic constriction. CONCLUSIONS: We conclude that IP3-R(2) do not contribute to the progression of DCM or pressure overload hypertrophy, despite increased expression and heightened generation of the ligand, Ins(1,4,5)P3.

Ritonavir stimulates foam cell formation by activating PKC.

Alpha-subtype protein kinase C (PKCα) is closely related to cardiovascular disease. Ritonavir (RTV), which is a human immunodeficiency virus (HIV) protease inhibitor, can induce atherosclerosis in a PKC-dependent manner. However, it remains unclear how RTV acts on PKCα to induce pathological phenotypes. In this study, we obtained mouse peritoneal macrophages from adult female Kunmin mice. The results of Oil Red O staining and immunofluorescence using confocal laser scanning microscope demonstrated that RTV could induce foam cell formation and plasma membrane translocation of PKCα like phorbol-12-myristate-13-acetate (PMA, a PKC activator). Computational modeling also exhibited similar docking of RTV and PMA to PKCα and similar patterns of hydrophobic interaction and hydrogen bond formation. Further in vitro kinase activity studies revealed that RTV could elevate PKC activity. These data provided insight into the PKC-dependent induction of atherosclerosis and useful information for more in-depth toxicity research of HIV protease inhibitor (PI). In addition, western blot analysis proved RTV also up-regulate PKCα expression, which may be related to its influence on estrogen responsiveness in target cells and needs further prove.

A liver-specific microRNA binds to a highly conserved RNA sequence of hepatitis B virus and negatively regulates viral gene expression and replication.

Regulated gene expression and progeny production are essential for persistent and chronic infection by human pathogens, such as hepatitis B virus (HBV), which affects >400 million people worldwide and is a major cause of liver disease. In this study, we provide the first direct evidence that a liver-specific microRNA, miR-122, binds to a highly conserved HBV pregenomic RNA sequence via base-pairing interactions and inhibits HBV gene expression and replication. The miR-122 target sequence is located at the coding region of the mRNA for the viral polymerase and the 3' untranslated region of the mRNA for the core protein. In cultured cells, HBV gene expression and replication reduces with increased expression of miR-122, and the expression of miR-122 decreases in the presence of HBV infection and replication. Furthermore, analyses of clinical samples demonstrated an inverse linear correlation in vivo between the miR-122 level and the viral loads in the peripheral blood mononuclear cells of HBV-positive patients. Our results suggest that miR-122 may down-regulate HBV replication by binding to the viral target sequence, contributing to the persistent/chronic infection of HBV, and that HBV-induced modulation of miR-122 expression may represent a mechanism to facilitate viral pathogenesis.

Hepatitis B virus induces a novel inflammation network involving three inflammatory factors, IL-29, IL-8, and cyclooxygenase-2.

Chronic inflammation induced by hepatitis B virus (HBV) is a major causative factor associated with the development of cirrhosis and hepatocellular carcinoma. In this study, we investigated the roles of three inflammatory factors, IL-8, IL-29 (or IFN-λ1), and cyclooxygenase-2 (COX-2), in HBV infection. We showed that the expression of IL-29, IL-8, and COX-2 genes was enhanced in HBV-infected patients or in HBV-expressing cells. In HBV-transfected human lymphocytes and hepatocytes, IL-29 activates the production of IL-8, which in turn enhances the expression of COX-2. In addition, COX-2 decreases the production of IL-8, which in turn attenuates the expression of IL-29. Thus, we proposed that HBV infection induces a novel inflammation cytokine network involving three inflammatory factors that regulate each other in the order IL-29/IL-8/COX-2, which involves positive regulation and negative feedback. In addition, we also demonstrated that COX-2 expression activated by IL-8 was mediated through CREB and C/EBP, which maintains the inflammatory environment associated with HBV infection. Finally, we showed that the ERK and the JNK signaling pathways were cooperatively involved in the regulation of COX-2. We also demonstrated that IL-29 inhibits HBV replication and that IL-8 attenuates the expression of IL-10R2 and the anti-HBV activity of IL-29, which favors the establishment of persistent viral infection. These new findings provide insights for our understanding of the mechanism by which inflammatory factors regulate each other in response to HBV infection.