👤 Wolf-Ferdinand Wieland

High-risk human papillomaviruses (HPV), particularly HPV16, are major causes of anogenital and oropharyngeal cancers. The HPV late promoter, P670 in the case of HPV16, is activated upon host cell differentiation and drives the expression of viral capsid proteins. While differentiation-specific host transcription factors have been implicated in regulating this promoter, the mechanism remains incompletely understood. HPV E2 proteins activate transcription by interacting with the host protein BRD4 (Bromodomain-containing protein 4). A biotin proximity ligation screen identified several novel E2 interactors, of which many overlap with the BRD4 interactome, suggesting BRD4 mediates a large fraction of these interactions. One such interactor, ZC3H4 (Zinc finger CCCH domain-containing protein 4), is known to restrict the expression of long non-coding RNAs, including enhancer and promoter upstream antisense RNAs (uaRNAs). E2 recruits ZC3H4 in a BRD4-dependent manner to specifically activate the P670 promoter in reporter assays. Supporting this, E2 and ZC3H4 co-localize in cells with high P670 activity. ZC3H4 is upregulated during differentiation, and its knockdown in differentiated HPV16- or HPV31-positive cells reduces late viral transcripts in an E2-BRD4-dependent manner. Interestingly, knockdown of ZC3H4 does not increase viral uaRNAs, suggesting that ZC3H4 does not enhance HPV late transcription by regulating viral antisense transcription. High-risk human papillomaviruses (HPVs), particularly HPV16, can cause anogenital and oropharyngeal cancers. HPV16 relies on the differentiation-dependent activation of its late promoter, P670, to produce capsid proteins. While host transcription factors contribute to this regulation, the mechanisms remain incompletely defined. Our findings reveal that the viral E2 protein collaborates with the host protein BRD4-a critical transcriptional regulator-to recruit other cellular partners, such as ZC3H4. Normally, ZC3H4 suppresses non-coding RNAs in cells, but HPV16 repurposes it via BRD4 to activate P670. This interaction intensifies in differentiated cells, where ZC3H4 levels rise, and disrupting ZC3H4 specifically blocks late viral gene expression without affecting antisense viral transcription. This highlights a unique, differentiation-dependent strategy HPV16 uses to hijack host machinery for its replication. Show less

Atherosclerosis is driven by an inflammatory process of the vascular wall. The novel orphan G-protein coupled receptor 5B of family C (GPRC5B) is involved in drosophila sugar and lipid metabolism as well as mice adipose tissue inflammation. Here, we investigated the role of GPRC5B in the pro-atherogenic mechanisms of hyperglycemia and vascular inflammation. Immortalized and primary endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) were used for stimulation with high glucose or different cytokines. Adenoviral- or plasmid-driven GPRC5B overexpression and siRNA-mediated knockdown were performed in these cells to analyze functional and mechanistic pathways of GPRC5B. In ECs and VSMCs, stimulation with high glucose, TNFα or LPS induced a significant upregulation of endogenous GPRC5B mRNA and protein levels. GPRC5B overexpression and knockdown increased and attenuated, respectively, the expression of the pro-inflammatory cytokines TNFα, IL-1β, IL-6 as well as the pro-atherogenic vascular adhesion molecules ICAM-1 and VCAM-1. Furthermore, the expression and activity of the metalloproteinase MMP-9, a component of atherosclerotic plaque stabilization, were significantly enhanced by GPRC5B overexpression. Mechanistically, GPRC5B increased the phosphorylation of ERK1/2 and activated NFκB through a direct interaction with the tyrosine kinase Fyn. Our findings demonstrate that GPRC5B is upregulated in response to high glucose and pro-inflammatory signaling. GPRC5B functionally modulates the inflammatory activity in cells of the vascular wall, suggesting a pro-atherogenic GPRC5B-dependent positive feedback loop via Fyn and NFκB. Thus, GPRC5B warrants further attention as a novel pharmacological target for the treatment of vascular inflammation and possibly atherogenesis. Show less

Inflammation is a major driver of cardiac remodeling. Cardiac fibroblasts play an integral role in cardiac inflammation, fibrosis and remodeling. The orphan G-protein-coupled-receptor 5B of family C (GPRC5B) has recently been shown to have pro-inflammatory effects in adipocytes via the NFκB-signaling-pathway. Here, we investigated whether GPRC5B is involved in myocardial inflammation and fibrosis. Using neonatal rat cardiac fibroblasts (NRCF) we show that the transcription and the expression of endogenous GPRC5B is induced by stimulation with TNFα and LPS as well as through cyclic mechanical stretch, while the principle pro-fibrotic factor TGFβ has no effect on the GPRC5B expression. Furthermore, we demonstrate that adenoviral overexpression and siRNA-mediated knockdown of GPRC5B in NRCF significantly alters the transcription level of the pro-inflammatory and pro-fibrotic cytokines TNFα, IL-1β, IL-6 and MCP-1, and extracellular matrix-degrading MMP-9 in vitro. Additionally, in adult GPRC5B-transgenic mice the protein expression of collagen-1A1 is decreased and the production of MMP-9 is increased, indicating remodeling of the extracellular matrix in vivo. Our data show that GPRC5B is up-regulated by inflammatory signals and mechanical stress in NRCF, while GPRC5B modulates the inflammatory response of cardiac fibroblasts and the degradation of extracellular matrix-proteins in the mice heart. Thus, our findings are the first to report a novel role of the orphan receptor GPRC5B in fibroblast-driven myocardial inflammation and cardiac remodeling. Show less

NALCN is a conserved cation channel, which conducts a permanent sodium leak current and regulates resting membrane potential and neuronal excitability. It is part of a large ion channel complex, the "NALCN channelosome", consisting of multiple proteins including UNC80 and UNC79. The predominant neuronal expression pattern and its function suggest an important role in neuronal function and disease. So far, biallelic NALCN and UNC80 variants have been described in a small number of individuals leading to infantile hypotonia, psychomotor retardation, and characteristic facies 1 (IHPRF1, OMIM 615419) and 2 (IHPRF2, OMIM 616801), respectively. Heterozygous de novo NALCN missense variants in the S5/S6 pore-forming segments lead to congenital contractures of the limbs and face, hypotonia, and developmental delay (CLIFAHDD, OMIM 616266) with some clinical overlap. In this study, we present detailed clinical information of 16 novel individuals with biallelic NALCN variants, 1 individual with a heterozygous de novo NALCN missense variant and an interesting clinical phenotype without contractures, and 12 individuals with biallelic UNC80 variants. We report for the first time a missense NALCN variant located in the predicted S6 pore-forming unit inherited in an autosomal-recessive manner leading to mild IHPRF1. We show evidence of clinical variability, especially among IHPRF1-affected individuals, and discuss differences between the IHPRF1- and IHPRF2 phenotypes. In summary, we provide a comprehensive overview of IHPRF1 and IHPRF2 phenotypes based on the largest cohort of individuals reported so far and provide additional insights into the clinical phenotypes of these neurodevelopmental diseases to help improve counseling of affected families. Show less

Recent studies have underlined the role of nuclear receptors in the involvement of prostate cancer signalling pathways. A total of 84 benign prostate hyperplasia (BPH), 84 low risk prostate cancer (LPC) and 64 advanced disease (APC) cases were sampled on a tissue microarray (TMA) and stained for retinoic acid receptor (RAR)-α, retionoid X receptor (RXR)-α, liver X receptor (LXR)-α, farnesoid X receptor (FXR) and proliferate-activated receptor gamma (PPAR)-γ and the (pro)-inflammatory molecules cyclooxygenase 2 (COX2), tumor necrosis factor (TNF)-α and inducible Nitric oxide synthase (iNOS) immunohistochemically. PPAR-γ expression in APC tissues was found to be significantly higher than that in LPC and BPH specimens (p<0.001). In contrast, RXR-a expression was significantly lower (p<0.001). COX2 staining demonstrated a trend towards overexpression in APC (p=0.025). No significant differences were found for RAR-α, iNOS and TNF-α expression. Staining of FXR and LXR was seen diffusely in the cytoplasm as well as in the nucleus, preventing sufficient evaluation by definition. This study provides the basis for applying PPAR-γ ligands clinically in treatment of APC. Show less