Sox2 and Lef-1 interact with Pitx2 to regulate incisor development and stem cell renewal.

Sox2 marks dental epithelial stem cells (DESCs) in both mammals and reptiles, and in this article we demonstrate several Sox2 transcriptional mechanisms that regulate dental stem cell fate and incisor growth. Conditional Sox2 deletion in the oral and dental epithelium results in severe craniofacial defects, including impaired dental stem cell proliferation, arrested incisor development and abnormal molar development. The murine incisor develops initially but is absorbed independently of apoptosis owing to a lack of progenitor cell proliferation and differentiation. Tamoxifen-induced inactivation of Sox2 demonstrates the requirement of Sox2 for maintenance of the DESCs in adult mice. Conditional overexpression of Lef-1 in mice increases DESC proliferation and creates a new labial cervical loop stem cell compartment, which produces rapidly growing long tusk-like incisors, and Lef-1 epithelial overexpression partially rescues the tooth arrest in Sox2 conditional knockout mice. Mechanistically, Pitx2 and Sox2 interact physically and regulate Lef-1, Pitx2 and Sox2 expression during development. Thus, we have uncovered a Pitx2-Sox2-Lef-1 transcriptional mechanism that regulates DESC homeostasis and dental development.

Wnt Signaling Regulates Airway Epithelial Stem Cells in Adult Murine Submucosal Glands.

Wnt signaling is required for lineage commitment of glandular stem cells (SCs) during tracheal submucosal gland (SMG) morphogenesis from the surface airway epithelium (SAE). Whether similar Wnt-dependent processes coordinate SC expansion in adult SMGs following airway injury remains unknown. We found that two Wnt-reporters in mice (BAT-gal and TCF/Lef:H2B-GFP) are coexpressed in actively cycling SCs of primordial glandular placodes and in a small subset of adult SMG progenitor cells that enter the cell cycle 24 hours following airway injury. At homeostasis, these Wnt reporters showed nonoverlapping cellular patterns of expression in the SAE and SMGs. Following tracheal injury, proliferation was accompanied by dynamic changes in Wnt-reporter activity and the analysis of 56 Wnt-related signaling genes revealed unique temporal changes in expression within proximal (gland-containing) and distal (gland-free) portions of the trachea. Wnt stimulation in vivo and in vitro promoted epithelial proliferation in both SMGs and the SAE. Interestingly, slowly cycling nucleotide label-retaining cells (LRCs) of SMGs were spatially positioned near clusters of BAT-gal positive serous tubules. Isolation and culture of tet-inducible H2B-GFP LRCs demonstrated that SMG LRCs were more proliferative than SAE LRCs and culture expanded SMG-derived progenitor cells outcompeted SAE-derived progenitors in regeneration of tracheal xenograft epithelium using a clonal analysis competition assay. SMG-derived progenitors were also multipotent for cell types in the SAE and formed gland-like structures in xenografts. These studies demonstrate the importance of Wnt signals in modulating SC phenotypes within tracheal niches and provide new insight into phenotypic differences of SMG and SAE SCs. Stem Cells 2016;34:2758-2771.

Conditional deletion of Sox17 reveals complex effects on uterine adenogenesis and function.

The importance of canonical Wnt signaling to murine uterine development is well established. Mouse models in which uterine-specific Wnt ligands, ß-catenin, or Lef1 are disrupted result in failure of postnatal endometrial gland development. Sox17 is a transcription factor characterized in numerous tissues as an antagonist of Wnt signaling. Thus, we hypothesized that conditional ablation of Sox17 would lead to hyperproliferation of endometrial glands in mice. Contrary to our prediction, disruption of Sox17 in epithelial and stromal compartments led to inhibition of endometrial adenogenesis and a loss of reproductive capacity. Epithelium-specific Sox17 disruption resulted in normal adenogenesis although reproductive capacity remained impaired. These findings suggest that non-epithelial, Sox17-positive cells are necessary for adenogenesis and that glands require Sox17 to properly function. To our knowledge, these findings are the first to implicate Sox17 in endometrial gland formation and reproductive success. The data presented herein underscore the importance of studying Sox17 in uterine homeostasis and function.

Microvessel density and p53 mutations in advanced-stage epithelial ovarian cancer.

We planned to determine the relationship between angiogenesis and p53 mutational status in advanced-stage epithelial ovarian cancer. Using 190 tumor samples from patients with stage III and IV ovarian cancer we performed p53 sequencing, immunohistochemistry, and CD31 microvessel density (MVD) determination. MVD was elevated in tumors with p53 null mutations compared to p53 missense mutation or no mutation. Disease recurrence was increased with higher MVD in both unadjusted and adjusted analyses. In adjusted analysis, p53 null mutation was associated with increased recurrence and worse overall survival. Worse overall survival and increased recurrence risk were also associated with the combination of CD31 MVD values >25 vessels/HPF and any p53 mutation. P53 mutation status and MVD may have prognostic significance in patients with advanced-stage ovarian cancer. Tumors with p53 null mutations are likely to be more vascular, contributing to decreased survival and increased recurrence probability.

The role of LEF1 in endometrial gland formation and carcinogenesis.

Endometrial carcinoma is the most common gynecologic cancer, yet the mechanisms underlying this disease process are poorly understood. We hypothesized that Lef1 is required for endometrial gland formation within the uterus and is overexpressed in endometrial cancer. Using Lef1 knockout (KO) mice, we compared uterine gland development to wild-type (WT) controls, with respect to both morphology and expression of the Lef1 targets, cyclin D1 and MMP7. We characterized the dynamics of Lef1 protein expression during gland development and the mouse estrus cycle, by immunostaining and Western blot. Finally, we investigated the roles of cyclin D1 and MMP7 in gland and cancer formation in the mouse, and assessed the relevance of Lef1 to human cancer by comparing expression levels in cancerous and normal endometrial tissues. Lef1 upregulation in mouse endometrium correlates with the proliferative stages of the estrus cycle and gland development during the neonatal period. WT mice endometrial glands began to develop by day 5 and were easily identified by day 9, whereas Lef1 KO mice endometrial glands had not developed by day 9 although the endometrial lining was intact. We found that during gland development cyclin D1 is elevated and localized to the gland buds, and that this requires the presence of Lef1. We also noted that Lef1 protein was expressed at higher levels in endometrial cancers within mice and humans when compared to normal endometrium. Our loss-of-function data indicate that Lef1 is required for the formation of endometrial glands in the mouse uterus. Lef1 protein elevation corresponds to gland formation during development, and varies cyclically with the mouse estrus cycle, in parallel with gland regeneration. Finally, Lef1 is overexpressed in human and mouse endometrial tumors, consistent with it playing a role in gland proliferation.

CGRP induction in cystic fibrosis airways alters the submucosal gland progenitor cell niche in mice.

In cystic fibrosis (CF), a lack of functional CF transmembrane conductance regulator (CFTR) chloride channels causes defective secretion by submucosal glands (SMGs), leading to persistent bacterial infection that damages airways and necessitates tissue repair. SMGs are also important niches for slow-cycling progenitor cells (SCPCs) in the proximal airways, which may be involved in disease-related airway repair. Here, we report that calcitonin gene-related peptide (CGRP) activates CFTR-dependent SMG secretions and that this signaling pathway is hyperactivated in CF human, pig, ferret, and mouse SMGs. Since CGRP-expressing neuroendocrine cells reside in bronchiolar SCPC niches, we hypothesized that the glandular SCPC niche may be dysfunctional in CF. Consistent with this hypothesis, CFTR-deficient mice failed to maintain glandular SCPCs following airway injury. In wild-type mice, CGRP levels increased following airway injury and functioned as an injury-induced mitogen that stimulated SMG progenitor cell proliferation in vivo and altered the proliferative potential of airway progenitors in vitro. Components of the receptor for CGRP (RAMP1 and CLR) were expressed in a very small subset of SCPCs, suggesting that CGRP indirectly stimulates SCPC proliferation in a non-cell-autonomous manner. These findings demonstrate that CGRP-dependent pathways for CFTR activation are abnormally upregulated in CF SMGs and that this sustained mitogenic signal alters properties of the SMG progenitor cell niche in CF airways. This discovery may have important implications for injury/repair mechanisms in the CF airway.

Regulation of surfactant protein D in the mouse female reproductive tract in vivo.

Surfactant protein D (SP-D) plays a role in innate immunity in the lung and is expressed at many other mucosal surfaces throughout the human body. In this study, we show that SP-D mRNA and protein are present in the murine female reproductive tract; i.e. in the vagina, cervix, uterus and oviduct. SP-D protein is primarily localized to epithelial cells lining the genital tract and is also present in secretory material within the lumen of the uterus and cervix. The levels of SP-D mRNA in the uterus vary by a factor of 10 during the estrous cycle with peak levels present at estrus and the lowest levels at diestrus. In contrast, SP-D mRNA levels in the lung do not change during the estrous cycle. Since SP-D is an innate host defense protein present in the mouse reproductive tract, we studied the influence of infection on SP-D levels in vivo. We found that Chlamydia muridarum infection caused an increase in the SP-D protein content of reproductive tract epithelial cells. These data are suggestive that SP-D may play a role in innate immunity in the female reproductive tract in vivo.

Wnt3a regulates Lef-1 expression during airway submucosal gland morphogenesis.

Regulation of the lymphoid enhancer factor 1 (Lef-1) transcription factor is important for the inductive formation of many epithelial-derived appendages including airway submucosal glands (SMGs). Although Wnts have been linked to developmental processes involving transcriptional activation of the Lef-1 protein, there is little in vivo information directly linking Wnts with the transcriptional regulation of the Lef-1 promoter. In the present study, we hypothesized that Wnt3a directly regulates Lef-1 gene expression required for SMG morphogenesis in mice. In support of this hypothesis, TOPGAL reporter mice demonstrated activation of beta-catenin/Tcf complexes during early phases of SMG development and immunolocalization studies confirmed abundant expression of Tcf4, but not Tcf1 or Tcf3, at this stage. ChIP analysis in primary airway epithelial cells revealed that Tcf4 associates with a known Wnt Responsive Region in the Lef-1 promoter and transfection of Cos-1 cells with dominant active beta-catenin and Tcf4 synergistically activated the Lef-1 promoter. Using Wnt3a deficient and Lef-1 promoter-GFP reporter mice, we also demonstrate that Wnt3a induces Lef-1 gene expression in newly forming SMG buds of mice and is required for the maintenance of gland bud growth. These findings provide the first in vivo evidence that Wnt3a can transcriptionally regulate the Lef-1 gene.

Surfactant proteins A and D enhance the phagocytosis of Chlamydia into THP-1 cells.

Chlamydiae are intracellular bacterial pathogens that infect mucosal surfaces, i.e., the epithelium of the lung, genital tract, and conjunctiva of the eye, as well as alveolar macrophages. In the present study, we show that pulmonary surfactant protein A (SP-A) and surfactant protein D (SP-D), lung collectins involved in innate host defense, enhance the phagocytosis of Chlamydia pneumoniae and Chlamydia trachomatis by THP-1 cells, a human monocyte/macrophage cell line. We also show that SP-A is able to aggregate both C. trachomatis and C. pneumoniae but that SP-D only aggregates C. pneumoniae. In addition, we found that after phagocytosis in the presence of SP-A, the number of viable C. trachomatis pathogens in the THP-1 cells 48 h later was increased approximately 3.5-fold. These findings suggest that SP-A and SP-D interact with chlamydial pathogens and enhance their phagocytosis into macrophages. In addition, the chlamydial pathogens internalized in the presence of collectins are able to grow and replicate in the THP-1 cells after phagocytosis.