IL-38 regulates intestinal stem cell homeostasis by inducing WNT signaling and beneficial IL-1ß secretion.

The IL-1 Family member IL-38 has been characterized primarily as an antiinflammatory cytokine in human and mouse models of systemic diseases. Here, we examined the role of IL-38 in the murine small intestine (SI). Immunostaining of SI revealed that IL-38 expression partially confines to intestinal stem cells. Cultures of intestinal organoids reveal IL-38 functions as a growth factor by increasing organoid size via inducing WNT3a. In contrast, organoids from IL-38-deficient mice develop more slowly. This reduction in size is likely due to the downregulation of intestinal stemness markers (i.e., Fzd5, Ephb2, and Olfm4) expression compared with wild-type organoids. The IL-38 binding to IL-1R6 and IL-1R9 is still a matter of debate. Therefore, to analyze the molecular mechanisms of IL-38 signaling, we also examined organoids from IL-1R9-deficient mice. Unexpectedly, these organoids, although significantly smaller than wild type, respond to IL-38, suggesting that IL-1R9 is not involved in IL-38 signaling in the stem cell crypt. Nevertheless, silencing of IL-1R6 disabled the organoid response to the growth property of IL-38, thus suggesting IL-1R6 as the main receptor used by IL-38 in the crypt compartment. In organoids from wild-type mice, IL-38 stimulation induced low concentrations of IL-1ß which contribute to organoid growth. However, high concentrations of IL-1ß have detrimental effects on the cultures that were prevented by treatment with recombinant IL-38. Overall, our data demonstrate an important regulatory function of IL-38 as a growth factor, and as an antiinflammatory molecule in the SI, maintaining homeostasis.

Y705 and S727 are required for the mitochondrial import and transcriptional activities of STAT3, and for regulation of stem cell proliferation.

The STAT3 transcription factor, acting both in the nucleus and mitochondria, maintains embryonic stem cell pluripotency and promotes their proliferation. In this work, using zebrafish, we determined in vivo that mitochondrial STAT3 regulates mtDNA transcription in embryonic and larval stem cell niches and that this activity affects their proliferation rates. As a result, we demonstrated that import of STAT3 inside mitochondria requires Y705 phosphorylation by Jak, whereas its mitochondrial transcriptional activity, as well as its effect on proliferation, depends on the MAPK target S727. These data were confirmed using mouse embryonic stem cells: although the Y705-mutated STAT3 cannot enter mitochondria, the S727 mutation does not affect import into the organelle and is responsible for STAT3-dependent mitochondrial transcription. Surprisingly, STAT3-dependent increase of mitochondrial transcription appears to be independent from STAT3 binding to STAT3-responsive elements. Finally, loss-of-function experiments, with chemical inhibition of the JAK/STAT3 pathway or genetic ablation of stat3 gene, demonstrated that STAT3 is also required for cell proliferation in the intestine of zebrafish.

Correction: Zanin et al. Analysis of Radiation Toxicity in Mammalian Cells Stably Transduced with Mitochondrial Stat3. Int. J. Mol. Sci. 2023, 24, 8232.

In the original publication [...].

Analysis of Radiation Toxicity in Mammalian Cells Stably Transduced with Mitochondrial Stat3.

A coordinated action between nuclear and mitochondrial activities is essential for a proper cellular response to genotoxic stress. Several nuclear transcription factors, including STAT3, translocate to mitochondria to exert mitochondrial function regulation; however, the role of mitochondrial STAT3 (mitoSTAT3) under stressed conditions is still poorly understood. In this study, we examined whether the stable expression of mitoSTAT3 wild-type or mutated at the conserved serine residue (Ser727), which is involved in the mitochondrial function of STAT3, can affect the DNA damage response to UVC radiation. To address this issue, we generated mammalian cells (NIH-3T3 and HCT-116 cells) stably transduced to express the mitochondrial-targeted Stat3 gene in its wild-type or Ser727 mutated forms. Our results show that cell proliferation is enhanced in mitoStat3-transduced cells under both non-stressed and stressed conditions. Once irradiated with UVC, cells expressing wild-type mitoSTAT3 showed the highest cell survival, which was associated with a significant decrease in cell death. Low levels of oxidative stress were detected in UVC-irradiated NIH-3T3 cells expressing mitoSTAT3 wild-type or serine-related dominant active form (Ser727D), confirming a role of mitochondrial STAT3 in minimizing oxidant cellular stress that provides an advantage for cell survival.

Zebrafish Mutant Lines Reveal the Interplay between nr3c1 and nr3c2 in the GC-Dependent Regulation of Gene Transcription.

Glucocorticoids mainly exert their biological functions through their cognate receptor, encoded by the nr3c1 gene. Here, we analysed the glucocorticoids mechanism of action taking advantage of the availability of different zebrafish mutant lines for their receptor. The differences in gene expression patterns between the zebrafish gr knock-out and the grs357 mutant line, in which a point mutation prevents binding of the receptor to the hormone-responsive elements, reveal an intricate network of GC-dependent transcription. Particularly, we show that Stat3 transcriptional activity mainly relies on glucocorticoid receptor GR tethering activity: several Stat3 target genes are induced upon glucocorticoid GC exposure both in wild type and in grs357/s357 larvae, but not in gr knock-out zebrafish. To understand the interplay between GC, their receptor, and the mineralocorticoid receptor, which is evolutionarily and structurally related to the GR, we generated an mr knock-out line and observed that several GC-target genes also need a functional mineralocorticoid receptor MR to be correctly transcribed. All in all, zebrafish mutants and transgenic models allow in vivo analysis of GR transcriptional activities and interactions with other transcription factors such as MR and Stat3 in an in-depth and rapid way.

Sex and Brain: The Role of Sex Chromosomes and Hormones in Brain Development and Parkinson's Disease.

Sex hormones and genes on the sex chromosomes are not only key factors in the regulation of sexual differentiation and reproduction but they are also deeply involved in brain homeostasis. Their action is crucial for the development of the brain, which presents different characteristics depending on the sex of individuals. The role of these players in the brain is fundamental in the maintenance of brain function during adulthood as well, thus being important also with respect to age-related neurodegenerative diseases. In this review, we explore the role of biological sex in the development of the brain and analyze its impact on the predisposition toward and the progression of neurodegenerative diseases. In particular, we focus on Parkinson's disease, a neurodegenerative disorder that has a higher incidence in the male population. We report how sex hormones and genes encoded by the sex chromosomes could protect from the disease or alternatively predispose toward its development. We finally underline the importance of considering sex when studying brain physiology and pathology in cellular and animal models in order to better understand disease etiology and develop novel tailored therapeutic strategies.

STAT3 and HIF1α cooperatively mediate the transcriptional and physiological responses to hypoxia.

STAT3 and HIF1α are two fundamental transcription factors involved in many merging processes, like angiogenesis, metabolism, and cell differentiation. Notably, under pathological conditions, the two factors have been shown to interact genetically, but both the molecular mechanisms underlying such interactions and their relevance under physiological conditions remain unclear. In mouse embryonic stem cells (ESCs) we manage to determine the specific subset of hypoxia-induced genes that need STAT3 to be properly transcribed and, among them, fundamental genes like Vegfa, Hk1, Hk2, Pfkp and Hilpda are worth mentioning. Unexpectedly, we also demonstrated that the absence of STAT3 does not affect the expression of Hif1α mRNA nor the stabilization of HIF1α protein, but the STAT3-driven regulation of the hypoxia-dependent subset of gene could rely on the physical interaction between STAT3 and HIF1α. To further elucidate the physiological roles of this STAT3 non-canonical nuclear activity, we used a CRISPR/Cas9 zebrafish stat3 knock-out line. Notably, hypoxia-related fluorescence of the hypoxia zebrafish reporter line (HRE:mCherry) cannot be induced when Stat3 is not active and, while Stat3 Y705 phosphorylation seems to have a pivotal role in this process, S727 does not affect the Stat3-dependent hypoxia response. Hypoxia is fundamental for vascularization, angiogenesis and immune cells mobilization; all processes that, surprisingly, cannot be induced by low oxygen levels when Stat3 is genetically ablated. All in all, here we report the specific STAT3/HIF1α-dependent subset of genes in vitro and, for the first time with an in vivo model, we determined some of the physiological roles of STAT3-hypoxia crosstalk.

The Roles of Post-Translational Modifications in STAT3 Biological Activities and Functions.

STAT3 is an important transcription factor that regulates cell growth and proliferation by regulating gene transcription of a plethora of genes. This protein also has many roles in cancer progression and several tumors such as prostate, lung, breast, and intestine cancers that are characterized by strong STAT3-dependent transcriptional activity. This protein is post-translationally modified in different ways according to cellular context and stimulus, and the same post-translational modification can have opposite effects in different cellular models. In this review, we describe the studies performed on the main modifications affecting the activity of STAT3: phosphorylation of tyrosine 705 and serine 727; acetylation of lysine 49, 87, 601, 615, 631, 685, 707, and 709; and methylation of lysine 49, 140, and 180. The extensive results obtained by different studies demonstrate that post-translational modifications drastically change STAT3 activities and that we need further analysis to properly elucidate all the functions of this multifaceted transcription factor.