FGFR2 is essential for salivary gland duct homeostasis and MAPK-dependent seromucous acinar cell differentiation.

Exocrine acinar cells in salivary glands (SG) are critical for oral health and loss of functional acinar cells is a major clinical challenge. Fibroblast growth factor receptors (FGFR) are essential for early development of multiple organs, including SG. However, the role of FGFR signaling in specific populations later in development and during acinar differentiation are unknown. Here, we use scRNAseq and conditional deletion of murine FGFRs in vivo to identify essential roles for FGFRs in craniofacial, early SG development and progenitor function during duct homeostasis. Importantly, we also discover that FGFR2 via MAPK signaling is critical for seromucous acinar differentiation and secretory gene expression, while FGFR1 is dispensable. We show that FGF7, expressed by myoepithelial cells (MEC), activates the FGFR2-dependent seromucous transcriptional program. Here, we propose a model where MEC-derived FGF7 drives seromucous acinar differentiation, providing a rationale for targeting FGFR2 signaling in regenerative therapies to restore acinar function.

FGFR2b is essential for salivary gland duct homeostasis and MAPK-dependent seromucous acinar cell differentiation.

Exocrine secretory acinar cells in salivary glands (SG) are critical for oral health and loss of functional acinar cells is a major clinical challenge. Fibroblast growth factor receptors (FGFR) are essential for early development of multiple organs, including SG. However, the role of FGFR signaling in specific epithelial SG populations later in development and during acinar differentiation are unknown. Here, we predicted FGFR dependence in specific populations using scRNAseq data and conditional mouse models to delete FGFRs in vivo. We identifed essential roles for FGFRs in craniofacial and early SG development, as well as progenitor function during duct homeostasis. Importantly, we discovered that FGFR2b was critical for seromucous and serous acinar cell differentiation and secretory gene expression (Bpifa2 and Lpo) via MAPK signaling, while FGFR1b was dispensable. We show that FGF7, expressed by myoepithelial cells (MEC), activated the FGFR2b-dependent seromucous transcriptional program. We propose a model where MEC-derived FGF7 drives seromucous acinar differentiaton, providing a rationale for targeting FGFR2b signaling in regenerative therapies to restore acinar function.

Weighted analysis of 2'-fucosyllactose, 3-fucosyllactose, lacto-N-tetraose, 3'-sialyllactose, and 6'-sialyllactose concentrations in human milk.

Over 150 human milk oligosaccharides (HMOs) have been identified and their concentrations in human milk vary depending on Secretor and Lewis blood group status, environmental and geographical factors, lactation stage, gestational period, and maternal health. Quantitation of HMOs in human milk has been the focus of numerous studies, however, comprehensive and weighted statistical analyses of their levels in human milk are lacking. Therefore, weighted means, standard deviations, medians, interquartile ranges, and 90th percentiles for 2'-fucosyllactose (2'-FL), 3-fucosyllactose (3-FL), lacto-N-tetraose (LNT), 3'-sialyllactose (3'-SL) and 6'-sialyllactose (6'-SL) were calculated using random sampling and the levels of these HMOs in human milk reported in the literature. Probability distributions of the reported levels were also constructed. Although the levels reported in the published studies varied, the weighted means for 2'-FL, 3-FL, LNT, 3'-SL, and 6'-SL were calculated to be 2.58, 0.57, 0.94, 0.28, and 0.39 g/L, respectively, which are consistent with those that have been previously determined in other systematic analyses. Likely due to the use of weighting, the 90th percentiles were greater than the 95% confidence limits that have been previously calculated. Our study therefore provides accurate and important statistical data to help support the level of appropriate HMO supplementation in infant formula.

Neurturin Gene Therapy Protects Parasympathetic Function to Prevent Irradiation-Induced Murine Salivary Gland Hypofunction.

Head and neck cancer patients treated with irradiation often present irreversible salivary gland hypofunction for which no conventional treatment exists. We recently showed that recombinant neurturin, a neurotrophic factor, improves epithelial regeneration of mouse salivary glands in ex vivo culture after irradiation by reducing apoptosis of parasympathetic neurons. Parasympathetic innervation is essential to maintain progenitor cells during gland development and for regeneration of adult glands. Here, we investigated whether a neurturin-expressing adenovirus could be used for gene therapy in vivo to protect parasympathetic neurons and prevent gland hypofunction after irradiation. First, ex vivo fetal salivary gland culture was used to compare the neurturin adenovirus with recombinant neurturin, showing they both improve growth after irradiation by reducing neuronal apoptosis and increasing innervation. Then, the neurturin adenovirus was delivered to mouse salivary glands in vivo, 24 hr before irradiation, and compared with a control adenovirus. The control-treated glands have ∼50% reduction in salivary flow 60 days post-irradiation, whereas neurturin-treated glands have similar flow to nonirradiated glands. Further, markers of parasympathetic function, including vesicular acetylcholine transporter, decreased with irradiation, but not with neurturin treatment. Our findings suggest that in vivo neurturin gene therapy prior to irradiation protects parasympathetic function and prevents irradiation-induced hypofunction.

PKCδ regulates integrin αVß3 expression and transformed growth of K-ras dependent lung cancer cells.

We have previously shown that Protein Kinase C delta (PKCδ) functions as a tumor promoter in non-small cell lung cancer (NSCLC), specifically in the context of K-ras addiction. Here we define a novel PKCδ -> integrin αVß3 ->Extracellular signal-Regulated Kinase (ERK) pathway that regulates the transformed growth of K-ras dependent NSCLC cells. To explore how PKCδ regulates tumorigenesis, we performed mRNA expression analysis in four KRAS mutant NSCLC cell lines that stably express scrambled shRNA or a PKCδ targeted shRNA. Analysis of PKCδ-dependent mRNA expression identified 3183 regulated genes, 210 of which were specifically regulated in K-ras dependent cells. Genes that regulate extracellular matrix and focal adhesion pathways were most highly represented in this later group. In particular, expression of the integrin pair, αVß3, was specifically reduced in K-ras dependent cells with depletion of PKCδ, and correlated with reduced ERK activation and reduced transformed growth as assayed by clonogenic survival. Re-expression of PKCδ restored ITGAV and ITGB3 mRNA expression, ERK activation and transformed growth, and this could be blocked by pretreatment with a αVß3 function-blocking antibody, demonstrating a requirement for integrin αVß3 downstream of PKCδ. Similarly, expression of integrin αV restored ERK activation and transformed growth in PKCδ depleted cells, and this could also be inhibited by pretreatment with PD98059.Our studies demonstrate an essential role for αVß3 and ERK signalingdownstream of PKCδ in regulating the survival of K-ras dependent NSCLC cells, and identify PKCδ as a novel therapeutic target for the subset of NSCLC patients with K-ras dependent tumors.

Luminal signaling: it's what's on the inside that counts.

During organogenesis, FGFs are diffusible communication signals that allow cells to coordinate morphogenesis and establish tissue architecture. Recently in Nature, Durdu et al. (2014) show that epithelial cell clusters secrete FGFs into a microlumen, restricting FGF localization so that participating cells coordinate differentiation and collective migration via luminal signaling.

Protein kinase C δ is a downstream effector of oncogenic K-ras in lung tumors.

Oncogenic activation of K-ras occurs commonly in non-small cell lung cancer (NSCLC), but strategies to therapeutically target this pathway have been challenging to develop. Information about downstream effectors of K-ras remains incomplete, and tractable targets are yet to be defined. In this study, we investigated the role of protein kinase C δ (PKCδ) in K-ras-dependent lung tumorigenesis by using a mouse carcinogen model and human NSCLC cells. The incidence of urethane-induced lung tumors was decreased by 69% in PKCδ-deficient knockout (δKO) mice compared with wild-type (δWT) mice. δKO tumors are smaller and showed reduced proliferation. DNA sequencing indicated that all δWT tumors had activating mutations in KRAS, whereas only 69% of δKO tumors did, suggesting that PKCδ acts as a tumor promoter downstream of oncogenic K-ras while acting as a tumor suppressor in other oncogenic contexts. Similar results were obtained in a panel of NSCLC cell lines with oncogenic K-ras but which differ in their dependence on K-ras for survival. RNA interference-mediated attenuation of PKCδ inhibited anchorage-independent growth, invasion, migration, and tumorigenesis in K-ras-dependent cells. These effects were associated with suppression of mitogen-activated protein kinase pathway activation. In contrast, PKCδ attenuation enhanced anchorage-independent growth, invasion, and migration in NSCLC cells that were either K-ras-independent or that had WT KRAS. Unexpectedly, our studies indicate that the function of PKCδ in tumor cells depends on a specific oncogenic context, as loss of PKCδ in NSCLC cells suppressed transformed growth only in cells dependent on oncogenic K-ras for proliferation and survival.