Molecular characterization of centriole assembly in ciliated epithelial cells

Ciliated epithelial cells have the unique ability to generate hundreds of centrioles during differentiation. We used centrosomal proteins as molecular markers in cultured mouse tracheal epithelial cells to understand this process. Most centrosomal proteins were up-regulated early in ciliogenesis, initially appearing in cytoplasmic foci and then incorporated into centrioles. Three candidate proteins were further characterized. The centrosomal component SAS-6 localized to basal bodies and the proximal region of the ciliary axoneme, and depletion of SAS-6 prevented centriole assembly. The intraflagellar transport component polaris localized to nascent centrioles before incorporation into cilia, and depletion of polaris blocked axoneme formation. The centriolar satellite component PCM-1 colocalized with centrosomal components in cytoplasmic granules surrounding nascent centrioles. Interfering with PCM-1 reduced the amount of centrosomal proteins at basal bodies but did not prevent centriole assembly. This system will help determine the mechanism of centriole formation in mammalian cells and how the limitation on centriole duplication is overcome in ciliated epithelial cells

HIF1A-Dependent Induction of Alveolar Epithelial PFKFB3 Dampens Acute Lung Injury

Acute lung injury (ALI) is a severe form of lung inflammation causing acute respiratory distress syndrome in patients. ALI pathogenesis is closely linked to uncontrolled alveolar inflammation. We hypothesize that specific enzymes of the glycolytic pathway could function as key regulators of alveolar inflammation. Therefore, we screened isolated alveolar epithelia from mice exposed to ALI induced by injurious ventilation to assess their metabolic responses. These studies pointed us toward a selective role for isoform 3 of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3). Pharmacologic inhibition or genetic deletion of Pfkfb3 in alveolar epithelia (Pfkfb3loxP/loxP SPC-ER-Cre+ mice) was associated with profound increases in ALI during injurious mechanical ventilation or acid instillation. Studies in genetic models linked Pfkfb3 expression and function to Hif1a. Not only did intratracheal pyruvate instillation reconstitute Pfkfb3loxP/loxP or Hif1aloxP/loxP SPC-ER-Cre+ mice, but pyruvate was also effective in ALI treatment of wild-type mice. Finally, proof-of-principle studies in human lung biopsies demonstrated increased PFKFB3 staining in injured lungs and colocalized PFKFB3 to alveolar epithelia. These studies reveal a specific role for PFKFB3 in counterbalancing alveolar inflammation and lay the groundwork for novel metabolic therapeutic approaches during ALI

Cyclin-dependent kinase control of motile ciliogenesis

Cycling cells maintain centriole number at precisely two per cell in part by limiting their duplication to S phase under the control of the cell cycle machinery. In contrast, postmitotic multiciliated cells (MCCs) uncouple centriole assembly from cell cycle progression and produce hundreds of centrioles in the absence of DNA replication to serve as basal bodies for motile cilia. Although some cell cycle regulators have previously been implicated in motile ciliogenesis, how the cell cycle machinery is employed to amplify centrioles is unclear. We use transgenic mice and primary airway epithelial cell culture to show that Cdk2, the kinase responsible for the G1 to S phase transition, is also required in MCCs to initiate motile ciliogenesis. While Cdk2 is coupled with cyclins E and A2 during cell division, cyclin A1 is required during ciliogenesis, contributing to an alternative regulatory landscape that facilitates centriole amplification without DNA replication

Planar Cell Polarity Signaling: The Developing Cell’s Compass

Cells of many tissues acquire cellular asymmetry to execute their physiologic functions. The planar cell polarity system, first characterized in Drosophila, is important for many of these events. Studies in Drosophila suggest that an upstream system breaks cellular symmetry by converting tissue gradients to subcellular asymmetry, whereas a downstream system amplifies subcellular asymmetry and communicates polarity between cells. In this review, we discuss apparent similarities and differences in the mechanism that controls PCP as it has been adapted to a broad variety of morphological cellular asymmetries in various organisms

VANGL2 regulates luminal epithelial organization and cell turnover in the mammary gland.

The VANGL family of planar cell polarity proteins is implicated in breast cancer however its function in mammary gland biology is unknown. Here, we utilized a panel of Vang1 and Vangl2 mouse alleles to examine the requirement of VANGL family members in the murine mammary gland. We show that Vang1CKOΔ/Δ glands display normal branching while Vangl2flox/flox and Vangl2Lp/Lp tissue exhibit several phenotypes. In MMTV-Cre;Vangl2flox/flox glands, cell turnover is reduced and lumens are narrowed. A Vangl2 missense mutation in the Vangl2Lp/Lp tissue leads to mammary anlage sprouting defects and deficient outgrowth with transplantation of anlage or secondary tissue fragments. In successful Vangl2Lp/Lp outgrowths, three morphological phenotypes are observed: distended ducts, supernumerary end buds, and ectopic acini. Layer specific defects are observed with loss of Vangl2 selectively in either basal or luminal layers of mammary cysts. Loss in the basal compartment inhibits cyst formation, but has the opposite effect in the luminal compartment. Candidate gene analysis on MMTV-Cre;Vangl2flox/flox and Vangl2Lp/Lp tissue reveals a significant reduction in Bmi1 expression, with overexpression of Bmi1 rescuing defects in Vangl2 knockdown cysts. Our results demonstrate that VANGL2 is necessary for normal mammary gland development and indicate differential functional requirements in basal versus luminal mammary compartments

Distinct Spatiotemporally Dynamic Wnt-Secreting Niches Regulate Proximal Airway Regeneration and Aging.

Our understanding of dynamic interactions between airway basal stem cells (ABSCs) and their signaling niches in homeostasis, injury, and aging remains elusive. Using transgenic mice and pharmacologic studies, we found that Wnt/β-catenin within ABSCs was essential for proliferation post-injury in vivo. ABSC-derived Wnt ligand production was dispensable for epithelial proliferation. Instead, the PDGFRα+ lineage in the intercartilaginous zone (ICZ) niche transiently secreted Wnt ligand necessary for ABSC proliferation. Strikingly, ABSC-derived Wnt ligand later drove early progenitor differentiation to ciliated cells. We discovered additional changes in aging, as glandular-like epithelial invaginations (GLEIs) derived from ABSCs emerged exclusively in the ICZ of aged mice and contributed to airway homeostasis and repair. Further, ABSC Wnt ligand secretion was necessary for GLEI formation, and constitutive activation of β-catenin in young mice induced their formation in vivo. Collectively, these data underscore multiple spatiotemporally dynamic Wnt-secreting niches that regulate functionally distinct phases of airway regeneration and aging

Prickle1 mutation causes planar cell polarity and directional cell migration defects associated with cardiac outflow tract anomalies and other structural birth defects

Planar cell polarity (PCP) is controlled by a conserved pathway that regulates directional cell behavior. Here, we show that mutant mice harboring a newly described mutation termed Beetlejuice (Bj) in Prickle1 (Pk1), a PCP component, exhibit developmental phenotypes involving cell polarity defects, including skeletal, cochlear and congenital cardiac anomalies. Bj mutants die neonatally with cardiac outflow tract (OFT) malalignment. This is associated with OFT shortening due to loss of polarized cell orientation and failure of second heart field cell intercalation mediating OFT lengthening. OFT myocardialization was disrupted with cardiomyocytes failing to align with the direction of cell invasion into the outflow cushions. The expression of genes mediating Wnt signaling was altered. Also noted were shortened but widened bile ducts and disruption in canonical Wnt signaling. Using an in vitro wound closure assay, we showed Bj mutant fibroblasts cannot establish polarized cell morphology or engage in directional cell migration, and their actin cytoskeleton failed to align with the direction of wound closure. Unexpectedly, Pk1 mutants exhibited primary and motile cilia defects. Given Bj mutant phenotypes are reminiscent of ciliopathies, these findings suggest Pk1 may also regulate ciliogenesis. Together these findings show Pk1 plays an essential role in regulating cell polarity and directional cell migration during development