CRLF3 in human cells

Various hematopoietic growth factors that regulate the production of blood cells mediate additional homeostatic functions in other non-hematopoietic tissues. Examples are erythropoietin (Epo), thrombopoietin (Tpo) and granulocyte colony-stimulating factor (GCSF), all of which are also expressed in mammalian nervous systems where they regulate development, differentiation and cell survival. Their receptors (EpoR, TpoR and GCSFR) belong to the class I family of cytokine type I receptors that possess the extracellular cytokine receptor homology domain and a WSXWS motif. Particularly erythropoietin and its receptor have been intensively studied with respect to clinical treatment of anemia, neuroprotective/regenerative properties in compromised nervous systems and enhancement of cognitive performance in mice and humans.
Another member of this cytokine receptor family, the cytokine receptor-like factor 3 (CRLF3), has recently been implicated within the production of thrombocytes (Bennett et al. 2022, https://doi.org/10.1182/blood.2021013113). Human CRLF3 contains the characteristic cytokine receptor motif (WSXWS), a single-pass transmembrane region and a Janus kinase docking site and is expressed in various tissues including the nervous system. Though its ligand has not been identified and signalling mechanisms are largely unknown, CRLF3 has been associated with the regulation of proliferation, differentiation and cell survival, similar to its structurally related receptors for other hematopoietic cytokines. In contrast to the other, vertebrate-specific members of its cytokine receptor family, CRLF3 is highly conserved and present in all major eumetazoan taxa including cnidarians, various invertebrates and vertebrates including humans. Characterization of CRLF3 in different species and identifying its endogenous “ancient” ligands in invertebrates (Epo and EV-3 are vertebrate-specific) will likely fuel the hypothesis of a single origin of class 1 helical cytokines and their receptors, despite low sequence similarity among these molecules.

We generated CRLF3-knockout lines from two independent fibroblast-derived human induced pluripotent stem cell (iPSC) lines by CRISPR/Cas9 gene editing. CRLF3-KO lines along with wild-type and isogenic control lines were differentiated into neurons and exposed to rotenone for apoptosis induction. EV-3 (a neuroprotective human Epo-splice variant that does not activate EpoR) rescued wild-type and isogenic control neurons from rotenone-induced cell death, whereas CRLF3-KO neurons were not protected. Exposure to rotenone and/or EV-3 affected the expression of pro-apoptotic (BAX, Caspase 3) and anti-apoptotic (BCL-2, CRLF3) genes differentially in wild-type, isogenic control and CRLF3-KO neurons.