The Role of KH-Type Splicing Regulatory Protein (KSRP) for Immune Functions and Tumorigenesis.

Post-transcriptional control of gene expression is one important mechanism that enables stringent and rapid modulation of cytokine, chemokines or growth factors expression, all relevant for immune or tumor cell function and communication. The RNA-binding protein KH-type splicing regulatory protein (KSRP) controls the mRNA stability of according genes by initiation of mRNA decay and inhibition of translation, and by enhancing the maturation of microRNAs. Therefore, KSRP plays a pivotal role in immune cell function and tumor progression. In this review, we summarize the current knowledge about KSRP with regard to the regulation of immunologically relevant targets, and the functional role of KSRP on immune responses and tumorigenesis. KSRP is involved in the control of myeloid hematopoiesis. Further, KSRP-mediated mRNA decay of pro-inflammatory factors is necessary to keep immune homeostasis. In case of infection, functional impairment of KSRP is important for the induction of robust immune responses. In this regard, KSRP seems to primarily dampen T helper cell 2 immune responses. In cancer, KSRP has often been associated with tumor growth and metastasis. In summary, aside of initiation of mRNA decay, the KSRP-mediated regulation of microRNA maturation seems to be especially important for its diverse biological functions, which warrants further in-depth examination.

Knockout of the KH-Type Splicing Regulatory Protein Drives Glomerulonephritis in MRL-Faslpr Mice.

KH-type splicing regulatory protein (KSRP) is an RNA-binding protein that promotes mRNA decay and thereby negatively regulates cytokine expression at the post-transcriptional level. Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by dysregulated cytokine expression causing multiple organ manifestations; MRL-Faslpr mice are an established mouse model to study lupus disease pathogenesis. To investigate the impact of KSRP on lupus disease progression, we generated KSRP-deficient MRL-Faslpr mice (MRL-Faslpr/KSRP-/- mice). In line with the predicted role of KSRP as a negative regulator of cytokine expression, lupus nephritis was augmented in MRL-Faslpr/KSRP-/- mice. Increased infiltration of immune cells, especially of IFN-γ producing T cells and macrophages, driven by enhanced expression of T cell-attracting chemokines and adhesion molecules, seems to be responsible for worsened kidney morphology. Reduced expression of the anti-inflammatory interleukin-1 receptor antagonist may be another reason for severe inflammation. The increase of FoxP3+ T cells detected in the kidney seems unable to dampen the massive kidney inflammation. Interestingly, lymphadenopathy was reduced in MRL-Faslpr/KSRP-/- mice. Altogether, KSRP appears to have a complex role in immune regulation; however, it is clearly able to ameliorate lupus nephritis.

The RNA-Binding Protein KSRP Modulates Cytokine Expression of CD4+ T Cells.

The KH-type splicing regulatory protein (KSRP) is a RNA-binding protein, which regulates the stability of many mRNAs encoding immune-relevant proteins. As KSRP regulates innate immune responses, for instance by the modulation of type I interferon mRNA stability, we were interested whether knockdown of the protein (KSRP-/-) interferes with T cell activation and polarization. Polyclonally stimulated KSRP-/- CD4+ T cells proliferated at a higher extent and higher frequency and expressed the activation marker CD25 more than wild-type T cells. In supernatants of stimulated KSRP-/- CD4+ T cells, levels of IL-5, IL-9, IL-10, and IL-13 were observed to be increased compared to those of the control group. KSRP-/- CD8+ T cells showed no altered proliferative capacity upon polyclonal stimulation, but supernatants contained lower levels of interferon-γ. Similar changes in the cytokine expression patterns were also detected in T cells derived from KSRP-/- mice undergoing arthritis induction indicative of a pathophysiological role of KSRP-dependent T cell polarization. We demonstrated the direct binding of KSRP to the 3' untranslated region of IL-13, IL-10, and IFN-γ mRNA in in vitro experiments. Moreover, since IL-4 mRNA decay was reduced in KSRP-/- CD4+ T cells, we identify KSRP as a negative regulator of IL-4 expression. These data indicate that overexpression of IL-4, which constitutes the primary inducer of Th2 polarization, may cause the Th2 bias of polyclonally stimulated KSRP-/- CD4+ T cells. This is the first report demonstrating that KSRP is involved in the regulation of T cell responses. We present strong evidence that T cells derived from KSRP-/- mice favor Th2-driven immune responses.

The KH-type splicing regulatory protein (KSRP) regulates type III interferon expression post-transcriptionally.

Type III interferons (IFNs) are the latest members of the IFN family. They play an important role in immune defense mechanisms, especially in antiviral responses at mucosal sites. Moreover, they control inflammatory reactions by modulating neutrophil and dendritic cell functions. Therefore, it is important to identify cellular mechanisms involved in the control of type III IFN expression. All IFN family members contain AU-rich elements (AREs) in the 3'-untranslated regions (3'-UTR) of their mRNAs that determine mRNA half-life and consequently the expressional level of these cytokines. mRNA stability is controlled by different proteins binding to these AREs leading to either stabilization or destabilization of the respective target mRNA. The KH-type splicing regulatory protein KSRP (also named KHSRP) is an important negative regulator of ARE-containing mRNAs. Here, we identify the interferon lambda 3 (IFNL3) mRNA as a new KSRP target by pull-down and immunoprecipitation experiments, as well as luciferase reporter gene assays. We characterize the KSRP-binding site in the IFNL3 3'-UTR and demonstrate that KSRP regulates the mRNA half-life of the IFNL3 transcript. In addition, we detect enhanced expression of IFNL3 mRNA in KSRP-/- mice, establishing a negative regulatory function of KSRP in type III IFN expression also in vivo Besides KSRP the RNA-binding protein AUF1 (AU-rich element RNA-binding protein 1) also seems to be involved in the regulation of type III IFN mRNA expression.

Inactivation of the KSRP gene modifies collagen antibody induced arthritis.

The KH type splicing regulatory protein (KSRP) is a nucleic acid binding protein, which negatively regulates the stability and/or translatability of many mRNA species encoding immune-relevant proteins. As KSRP is expressed in immune cells including T and B cells, neutrophils, macrophages and dendritic cells, we wanted to analyze its importance for the development of autoimmune diseases. We chose collagen antibody-induced arthritis (CAIA) as an appropriate autoimmune disease mouse model in which neutrophils and macrophages constitute the main effector cell populations. We compared arthritis induction in wild type (WT) and KSRP-/- mice and paws were taken for histological sections and qPCR analysis. Furthermore, we determined the frequencies of spleen immune cells by flow cytometry. Cytokine levels in spleen cell supernatants were determined by cytometric bead array analyses (CBA). After CAIA induction we unexpectedly observed in WT animals much stronger swelling of the paws than in KSRP-/- mice. In accordance, histological staining of paw sections of KSRP-/- animals revealed much lower frequencies of infiltrating immune cells in the joints compared to WT animals. Furthermore, CAIA-treatment resulted in reduced expression of several inflammatory factors (like CXCL-1, iNOS, TNF-α and S100A8) as well as immune cell marker genes (e.g. LFA-1, CD68, Ly6G) in the joints of KSRP-/- mice. Spleen cells of KSRP-/- mice showed lower frequencies of myeloid cells. On cytokine level IFN-γ production was increased in spleen cells of KSRP-/- mice compared to WT samples. These data surprisingly suggest that the absence of KSRP protects against the induction of inflammatory arthritis.

Inhibitors of ß-catenin affect the immuno-phenotype and functions of dendritic cells in an inhibitor-specific manner.

Many tumors are characterized by mutation-induced constitutive activation of ß-catenin which promotes tumor growth and survival. Consequently, the development of specific ß-catenin inhibitors for tumor therapy has come into the focus of drug development. ß-Catenin was also shown to contribute to the tolerance-promoting function of unstimulated dendritic cells (DCs). In response to activation, DCs acquire potent T cell stimulatory capacity and induce profound tumor antigen-specific immune responses. Here we asked for effects of pre-clinically established ß-catenin inhibitors (CCT-031374, iCRT-5, PNU-75654) on mouse bone marrow-derived (BM)DCs. All three inhibitors moderately increased surface expression of MHCII, CD80, and CD86 on unstimulated DCs, but had no enhancing effect on their capacity to stimulate the proliferation of ovalbumin (OVA) specific CD4(+) T cells. CCT-031374 interfered with upregulation of costimulators (CD40, CD86) and cytokines (IL-1ß, TNF-α, IL-6, IL-10, IL-12) by LPS-stimulated DCs. Accordingly, this DC population displayed an impaired CD4(+) T cell stimulatory activity. iCRT-5 and PNU-75654 had no detrimental effects on the immuno-phenotype of stimulated DCs. Hence, DCs treated with iCRT-5 in the course of stimulation exerted comparably strong T cell proliferation as did control DCs. In contrast, DCs stimulated in the presence of PNU-75654 induced less T cell proliferation than the control population despite enhanced uptake and processing of OVA. Our findings suggest that the differential effects of ß-catenin inhibitors on stimulated DCs reflect off target effects. Concerning potential application of ß-catenin inhibitors for tumor therapy, iCRT-5 may be most beneficial, since it did not exert detrimental effects on stimulated DCs.