Renal cystic diseases are a leading cause of renal failure. Mutations associated with renal cystic diseases reside in genes encoding proteins that localize to primary cilia. These cystoproteins can disrupt ciliary structure or cilia-mediated signaling, although molecular mechanisms connecting cilia function to renal cystogenesis remain unclear. The ciliary gene, Thm1(Ttc21b), negatively regulates Hedgehog signaling and is most commonly mutated in ciliopathies. We report that loss of murine Thm1 causes cystic kidney disease, with persistent proliferation of renal cells, elevated cAMP levels, and enhanced expression of Hedgehog signaling genes. Notably, the cAMP-mediated cystogenic potential of Thm1-null kidney explants was reduced by genetically deleting Gli2, a major transcriptional activator of the Hedgehog pathway, or by culturing with small molecule Hedgehog inhibitors. These Hedgehog inhibitors acted independently of protein kinase A and Wnt inhibitors. Furthermore, simultaneous deletion of Gli2 attenuated the renal cystic disease associated with deletion of Thm1. Finally, transcripts of Hedgehog target genes increased in cystic kidneys of two other orthologous mouse mutants, jck and Pkd1, and Hedgehog inhibitors reduced cystogenesis in jck and Pkd1 cultured kidneys. Thus, enhanced Hedgehog activity may have a general role in renal cystogenesis and thereby present a novel therapeutic target.
Highlights d Epigenetic and gene expression profiles define molecular subtypes of uterine fibroids d DNA hypomethylation in the HMGA2 gene body is consistent with activation of the gene d HOXA gene cluster expression displays a more posterior pattern in uterine fibroids d Homeotic transformation appears to play a role in fibroid biology
Primary cilia extend from the plasma membrane of most vertebrate cells and mediate signaling pathways. Ciliary dysfunction underlies ciliopathies, which are genetic syndromes that manifest multiple clinical features, including renal cystic disease and obesity. THM1 (also termed TTC21B or IFT139) encodes a component of the intraflagellar transport-A complex and mutations in THM1 have been identified in 5% of individuals with ciliopathies. Consistent with this, deletion of murine Thm1 during late embryonic development results in cystic kidney disease. Here, we report that deletion of murine Thm1 during adulthood results in obesity, diabetes, hypertension and fatty liver disease, with gender differences in susceptibility to weight gain and metabolic dysfunction. Pair-feeding of Thm1 conditional knock-out mice relative to control littermates prevented the obesity and related disorders, indicating that hyperphagia caused the obese phenotype. Thm1 ablation resulted in increased localization of adenylyl cyclase III in primary cilia that were shortened, with bulbous distal tips on neurons of the hypothalamic arcuate nucleus, an integrative center for signals that regulate feeding and activity. In pre-obese Thm1 conditional knock-out mice, expression of anorexogenic pro-opiomelanocortin (Pomc) was decreased by 50% in the arcuate nucleus, which likely caused the hyperphagia. Fasting of Thm1 conditional knock-out mice did not alter Pomc nor orexogenic agouti-related neuropeptide (Agrp) expression, suggesting impaired sensing of changes in peripheral signals. Together, these data indicate that the Thm1-mutant ciliary defect diminishes sensitivity to feeding signals, which alters appetite regulation and leads to hyperphagia, obesity and metabolic disease.
STUDY QUESTION Can endometrial stromal stem/progenitor cell markers, SUSD2 and CD146/CD140b, enrich for human myometrial and fibroid stem/progenitor cells? SUMMARY ANSWER SUSD2 enriches for myometrial and fibroid cells that have mesenchymal stem cell (MSC) characteristics and can also be induced to decidualise. WHAT IS KNOWN ALREADY Mesenchymal stem-like cells have been separately characterised in the endometrial stroma and myometrium and may contribute to diseases in their respective tissues. STUDY DESIGN, SIZE, DURATION Normal myometrium, fibroids and endometrium were collected from hysterectomies with informed consent. Primary cells or tissues were used from at least three patient samples for each experiment. PARTICIPANTS/MATERIALS, SETTING, METHODS Flow cytometry, immunohistochemistry and immunofluorescence were used to characterise tissues. In vitro colony formation in normoxic and hypoxic conditions, MSC lineage differentiation (osteogenic and adipogenic) and decidualisation were used to assess stem cell activity. Xenotransplantation into immunocompromised mice was used to determine in vivo stem-like activity. Endpoint measures included quantitative PCR, colony formation, trichrome, Oil Red O and alkaline phosphatase activity staining. MAIN RESULTS AND THE ROLE OF CHANCE CD146+CD140b+ and/or SUSD2+ myometrial and fibroid cells were located in the perivascular region and formed more colonies in vitro compared to control cells and differentiated down adipogenic and osteogenic mesenchymal lineages in vitro. SUSD2+ myometrial cells had greater in vitro decidualisation potential, and SUSD2+ fibroid cells formed larger tumours in vivo compared to control cells. LARGE-SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION Markers used in this study enrich for cells with stem/progenitor cell activity; however, they do not distinguish stem from progenitor cells. SUSD2+ myometrial cells express markers of decidualisation when treated in vitro, but in vivo assays are needed to fully demonstration their ability to decidualise. WIDER IMPLICATIONS OF THE FINDINGS These results suggest a possible common MSC for the endometrial stroma and myometrium, which could be the tumour-initiating cell for uterine fibroids. STUDY FUNDING/COMPETING INTEREST(S) These studies were supported by NIH grants to JMT (R01OD012206) and to ALP (F32HD081856). The authors certify that we have no conflicts of interest to disclose.
Uterine remodeling during pregnancy is a fundamental, dynamic process required for successful propagation of eutherian species. The uterus can increase in size up to 40-fold during pregnancy, which is largely attributed to expansion of the myometrium by hyperplasia and hypertrophy. After pregnancy, the uterus repairs the remodeled or ''damaged'' tissue during uterine involution (INV). Little is known about this repair process, particularly the role of mesenchymal stem/progenitor cells. The objective of this study was to identify and characterize putative mesenchymal stem/progenitor cells in the murine myometrium using a combination of label retention and mesenchymal stem cell (MSC) marker expression and a pregnancy and uterine INV model. Tet-off transgenic mice with the Crelox system were used to specifically label mesenchymal cells (ie, myometrial and endometrial stromal cells) within the uterus while avoiding other cell types (eg, epithelial, immune, and endothelial cells) to identify slowly dividing cells and assess their stem cell qualities. We identified myometrial label-retaining cells (LRCs) that persisted for at least 3 months, expressed CD146 and CD140b (MSC markers), and proliferated at a higher rate during uterine INV compared with nonlabeled cells. The LRCs did not appear to express either estrogen receptor alpha or progesterone receptor, nor did the number of LRCs change at different estrous stages or in response to exogenous estradiol or progesterone administration, suggesting that LRCs were not involved in normal estrous cycling. The results from this study provide important insight into putative stem/progenitor cells in the myometrium and their possible role in uterine physiology.
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