Reduced serum testosterone (T), or hypogonadism, affects millions of men and is associated with many pathologies, including infertility, cardiovascular diseases, metabolic syndrome, and decreased libido and sexual function. Administering T-replacement therapy (TRT) reverses many of the symptoms associated with low T levels. However, TRT is linked to side effects such as infertility and increased risk of prostate cancer and cardiovascular diseases. Thus, there is a need to obtain T-producing cells that could be used to treat hypogonadism via transplantation and reestablishment of T-producing cell lineages in the body. T is synthesized by Leydig cells (LCs), proposed to derive from mesenchymal cells of mesonephric origin. Although mesenchymal cells have been successfully induced into LCs, the limited source and possible trauma to donors hinders their application to clinical therapies. Alternatively, human induced pluripotent stem cells (hiPSCs), which are expandable in culture and have the potential to differentiate into all somatic cell types, have become the emerging source of autologous cell therapies. We have successfully induced the differentiation of hiPSCs into either human Leydig-like (hLLCs) or adrenal-like cells (hALCs) using chemically defined culture conditions. Factors critical for the development of LCs were added to both culture systems. hLLCs expressed all steroidogenic genes and proteins important for T biosynthesis, synthesized T rather than cortisol, secreted steroid hormones in response to dibutyryl-cAMP and 22(R)-hydroxycholesterol, and displayed ultrastructural features resembling LCs. By contrast, hALCs synthesized cortisol rather than T. The success in generating hiPSC-derived hLLCs with broad human LC (hLC) features supports the potential for hiPSC-based hLC regeneration.

Animals utilize conserved mechanisms to regulate oxidative stress. The C. elegans SKN-1 protein is homologous to the vertebrate Nrf (NF-E2-related factor) family of cap ’n’ collar (CnC) transcription factors and functions as a core regulator of xenobiotic and oxidative stress responses. The WD40 repeat-containing protein WDR-23 is a key negative regulator of SKN-1 activity. We previously found that the oxidative stress induced by excess iodide can be relieved by loss of function in the BLI-3/TSP-15/DOXA-1 dual oxidase complex. To further understand the molecular mechanism of this process, we screened for new mutants that can survive in excess iodide and identified gain-of-function mutations in skn-1 and loss-of-function mutations in wdr-23. The SKN-1C isoform functions in the hypodermis to affect animal’s response to excess iodide, while the SKN-1A isoform appears to play a minor role. wdr-23(lf) can interact with bli-3 mutations in a manner different from skn-1(gf). Transcriptome studies suggest that excess iodide causes developmental arrest largely independent of changes in gene expression, and wdr-23(lf) could affect the expression of a subset of genes by a mechanism different from SKN-1 activation. We propose that WDR-23 and SKN-1 coordinate with the BLI-3/TSP-15/DOXA-1 dual oxidase complex in response to iodide-triggered oxidative stress.

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Oxidative stress response is a fundamental biological process mediated by conserved mechanisms. The identities and functions of some key regulators remain unknown. Here, we report a novel role of C. elegans casein kinase 1 gamma CSNK-1 (also known as CK1γ or CSNK1G) in regulating oxidative stress response and ROS levels. csnk-1 interacted with the bli-3/tsp-15/doxa-1 NADPH dual oxidase genes via genetic nonallelic noncomplementation to affect C. elegans survival in oxidative stress. The genetic interaction was supported by specific biochemical interactions between DOXA-1 and CSNK-1 and potentially between their human orthologs DUOXA2 and CSNK1G2. Consistently, CSNK-1 was required for normal ROS levels in C. elegans. CSNK1G2 and DUOXA2 each can promote ROS levels in human cells, effects that were suppressed by a small molecule casein kinase 1 inhibitor. We also detected genetic interactions between csnk-1 and skn-1 Nrf2 in oxidative stress response. Together, we propose that CSNK-1 CSNK1G defines a novel conserved regulatory mechanism for ROS homeostasis.

Animals utilize associated pathways to elicit responses to oxidative stress and infection. The molecular mechanisms coordinating these pathways remain unclear. Here, using C. elegans we identified the highly conserved casein kinase 1 gamma CSNK-1 (also known as CK1g or CSNK1G), as a key regulator of these processes. csnk-1 interacted with the bli-3/tsp-15/doxa-1 dual oxidase genes by nonallelic noncomplementation to negatively regulate animal survival in excess iodide, an oxidative stressor. A conserved interaction was detected between DOXA-1 and CSNK-1 and between their human homologs DUOXA2 and CSNK1G2. csnk-1 deficiency resulted in upregulated expression of innate immunity genes and increased animal survival in the pathogenic Pseudomonas aeruginosa PA14. Phosphoproteomic analyses identified decreased phosphorylation of key innate immunity regulators NSY-1 MAPKKK and LIN-45 Raf in csnk-1(lf) mutants. Indeed, NSY-1 and LIN-45 pathways were required for the increased survival of csnk-1-deficient animals in PA14. Further analyses suggest that CSNK-1 and SKN-1 Nrf2 might act in parallel to regulate oxidative stress response. Together, we propose that CSNK-1 CSNK1G plays a novel pivotal role in integrating animal’s responses to oxidative stress and pathogens.