1Universitätsklinikum Hamburg-Eppendorf Universitäres Herzzentrum, Klinik für Kardiologie Hamburg, Deutschland; 2Universitäres Herz- und Gefäßzentrum Hamburg Allgemeine und Interventionelle Kardiologie Hamburg, Deutschland; 3Universitäres Herz- und Gefäßzentrum Hamburg Klinik für Kardiologie Hamburg, Deutschland; 4Universitätsklinikum Hamburg-Eppendorf Universitäres Herzzentrum Hamburg, Deutschland
Background
Hypertension is one of the major contributors to cardiovascular diseases. While increasing evidence indicates an important role of immunity in the pathogenesis of hypertension, the underlying molecular
mechanisms are still not fully understood. Neopterin is a novel marker of immune activation produced by activated monocytes/macrophages as a by-product of the tetrahydrobiopterin (BH4) biosynthesis
through the rate-limiting enzyme GTP cyclohydrolase (GCH1). GCH1 activity is transcriptionally regulated by the IFNγ-mediated JAK/STAT1 pathway. BH4 is involved in production of nitric oxide and
its bioavailability is crucial for vascular tone regulation. Neopterin is implicated in atherosclerosis, is associated with the progression of heart failure disease and correlates closely with vascular
dysfunctions in hypertensive patients.
Previously, we identified the transcript encoding CRIP1 (Cysteine-rich protein 1) in human monocytes to be strongly associated with blood pressure changes. Furthermore, CRIP1 expression is influenced
by the pro-hypertensive factor Angiotensin II in splenic and circulating monocytes of hypertensive mice. Our recent data indicate an inverse correlation between CRIP1 mRNA expression in human
monocytes and serum levels of neopterin. So far, the function of CRIP1 the IFNγ-mediated Neopterin/BH4 production is unknown.
Aim
This project aimed to elucidate the involvement of CRIP1 in the IFNγ-mediated JAK/STAT1 pathway, which leads to the transcriptional activation of GCH1 and thus to the production of neopterin and BH4.
Methods
A stable shRNA-mediated downregulation of CRIP1 by 70% was generated in the monocytic cell line THP1. ShCRIP1-THP1 cells were stimulated with IFNγ at several time points (1, 6, 12 and 24 h). The
protein expression of CRIP1 and GCH1 was analysed by immunoblot. Neopterin/BH4 were measured in cell lysates and supernatants using corresponding ELISAs (B.R.A.H.M.S/Novus Biological).
Immunoblotting was used for investigation of the IFNγ-mediated JAK/STAT1 pathway.
Results
Results
Downregulation of CRIP1 leads to a significantly reduced GCH1 protein expression by 98.56% (p<0.0002), deregulated release and cellular content of BH4 and almost completely abolished IFNγ-induced production of neopterin by 83.77% (p<0.0001, ⴄ=0.47) upon 24h IFNγ stimulation. The downregulation of CRIP1 had no obvious effect on the protein expression of IFNγ receptor. However, a remarkable decrease in the protein levels STAT1 by 71.86% (p<0.0001) was observed. In addition, downregulated CRIP1 expression resulted in a substantial 50.16% increase in the phosphorylation of
STAT1 at the Y701 residue following 24h IFNγ treatment (p = 0.0004).
Conclusion
This work demonstrates a so far unknown connection between CRIP1, IFNγ-mediated JAK/STAT1 pathway, GCH1 expression and neopterin production, suggesting a considerable role of
Conclusion
This work demonstrates a so far unknown connection between CRIP1, IFNγ-mediated JAK/STAT1 pathway, GCH1 expression and neopterin production, suggesting a considerable role of
CRIP1 in pro-inflammatory conditions that are implicated in the pathophysiology of hypertension and cardiovascular diseases.