Abstract. The secreted polypeptide transforming growth factor-/3 (TGF-fl) exerts its multiple activities through type I and II cell surface receptors. In epithelial cells, activation of the TGF-fl signal transduction pathways leads to inhibition of cell proliferation and an increase in extracellular matrix production. TGF-/3 is widely expressed during development and its biological activity has been implicated in epithelial-mesenchymal interactions, e.g., in branching morphogenesis of the lung, kidney, and mammary gland, and in inductive events between mammary epithelium and stroma.In the present study, we investigated the effects of TGF-/3 on mouse mammary epithelial cells in vitro. TGF-/3 reversibly induced an alteration in the differentiation of normal mammary epithelial NMuMG cells from epithelial to fibroblastic phenotype. The change in cell morphology correlated with (a) decreased expression of the epithelial markers E-cadherin, ZO-1, and desmoplakin I and II; (b) increased expression of mesenchymal markers, such as fibronectin; and (c) a fibroblast-like reorganization of actin fibers. This phenotypic differentiation displays the hallmarks of an epithelial to mesenchymal transdifferentiation event.Since NMuMG cells make high levels of the type I TGF-fl receptor Tsk7L, yet lack expression of the ALK-5/R4 type I receptor which has been reported to mediate TGF-/~ responsiveness, we evaluated the role of the Tsk7L receptor in TGF-~-mediated transdifferentiation. We generated NMuMG cells that stably overexpress a truncated Tsk7L type I receptor that lacks most of the cytoplasmic kinase domain, thus function as a dominant negative mutant. These transfected cells no longer underwent epithelial to mesenchymal morphological change upon exposure to TGF-fl, yet still displayed some TGF-fl-mediated responses.We conclude that TGF-fl has the ability to modulate E-cadherin expression and induce a reversible epithelial to mesenchymal transdifferentiation in epithelial cells. Unlike other transdifferentiating growth factors, such as bFGF and HGF, these changes are accompanied by growth inhibition. Our results also implicate the Tsk7L type I receptor as mediating the TGF-~-induced epithelial to mesenchymal transition.

Since the discovery that epidermal growth factor (EGF) can accelerate opening of the eyelids, the EGF receptor (EGF-R) has been extensively studied and is now considered to be a prototype tyrosine kinase receptor. Binding of EGF or of transforming growth factor-alpha (TGF-alpha) or other related factors activates the receptor and induces cell proliferation and differentiation. Although it is not found on haematopoietic cells, the EGF-R is widely expressed in mammals and has been implicated in various stages of embryonic development. Here we investigate the developmental and physiological roles of this receptor and its ligands by inactivating the gene encoding EGF-R. We find that EGF-R-/- mice survive for up to 8 days after birth and suffer from impaired epithelial development in several organs, including skin, lung and gastrointestinal tract.

Craniofacial malformations are among the most frequent congenital birth defects in humans; cleft palate, that is inadequate fusion of the palatal shelves, occurs with an annual incidence of 1 in 700 to 1 in 1,000 live births among individuals of European descent. The secondary palate arises as bilateral outgrowths from the maxillary processes, and its formation depends on the coordinated development of craniofacial structures including the Meckel's cartilage and the mandible. Cleft lip and palate syndromes in humans are associated with polymorphisms in the gene (TGFA) encoding transforming growth factor-alpha (TGF-alpha), an epidermal growth factor receptor (EGFR) ligand made by most epithelia. Here we have characterized craniofacial development in Egfr-deficient (Egfr-/-) mice. Newborn Egfr-/- mice have facial mediolateral defects including narrow, elongated snouts, underdeveloped lower jaw and a high incidence of cleft palate. Palatal shelf explants from Egfr-/- mice fused, but frequently had residual epithelium in the midline. In addition, morphogenesis of Meckel's cartilage was deficient in cultured mandibular processes from Egfr-/- embryos. The secretion of matrix metalloproteinases (MMPs) was diminished in Egfr-/- explants, consistent with the ability of EGF to increase MMP secretion and with the decreased MMP expression caused by inhibition of Egfr signalling in wild-type explants. Accordingly, inactivation of MMPs in wild-type explants phenocopied the defective morphology of Meckel's cartilage seen in Egfr-/- explants. Our results indicate that EGFR signalling is necessary for normal craniofacial development and that its role is mediated in part by its downstream targets, the MMPs, and may explain the genetic correlation of human cleft palate with polymorphisms in TGFA.

Transforming growth factor-beta (TGF-beta) is the prototype for a family of extracellular polypeptides that affect cell proliferation and differentiation, and tissue morphogenesis. TGF-beta signalling is mediated by two types of serine/threonine kinase receptors, the type I and II receptors, which are able to form a heteromeric complex. No cytoplasmic proteins that associate with these receptors in vivo, or are their kinase targets, have yet been described. We have now identified a WD-domain-containing protein, TRIP-1, which specifically associates with the type II TGF-beta receptor in a kinase-dependent way. TRIP-1 does not interact with the type II activin or type I receptors, but associates with the heteromeric TGF-beta receptor complex. TRIP-1 is phosphorylated on serine and threonine by the receptor kinase, strongly suggesting that it has a role in TGF-beta signalling. This is supported by coexpression of TRIP-1 and type II receptor during development. The existence of TRIP-1 homologues in plant and yeast suggests a conserved function in all eukaryotes.

EGF-R regulates cell proliferation, migration, and invasion in fibroblasts. However, the connection of EGF-R with downstream signaling pathways mediating these responses has remained elusive. Here we provide genetic and biochemical evidence that EGF-R- and AP-1-mediated signals are required for MMP expression and collagen contraction in fibroblasts. In EGF-R (-/-) mouse embryonal fibroblasts, basal and inducible expression of several MMPs, including MMP-2, -3, and -14 is impaired in comparison to wild-type counterparts. The loss of MMP expression is associated with a suppression of EGF-induced Erk and Jnk activities, and AP-1 DNA-binding and transactivation capacities. While inhibition of Jnk mainly prevents EGF-induced phosphorylation of c-Jun, inhibition of Erk pathway suppresses both the expression and phosphorylation of c-Jun and c-Fos proteins. Moreover, the expression of MMP-3 and -14, and collagen contraction is partially prevented by Mek/Erk and Jnk inhibitors. However, Jnk inhibitor also suppresses cell growth independently of EGF-R activity. The central role of AP-1 as a mediator of EGF-R signaling in fibroblasts is emphasized by the finding that expression of a dominant negative c-Jun downregulates the expression of MMP-3. Conversely, expression of a constitutively active Mek1 can induce MMP-3 expression independently of upstream signals. The results indicate that ERK pathway and AP-1 are downstream effectors of the EGF-R-mediated MMP-3 expression and collagen contraction in fibroblasts.

Finding new causes of monogenic diabetes helps understand glycaemic regulation in humans. To find novel genetic causes of maturity-onset diabetes of the young (MODY), we sequenced MODY cases with unknown aetiology and compared variant frequencies to large public databases. From 36 European patients, we identify two probands with novel RFX6 heterozygous nonsense variants. RFX6 protein truncating variants are enriched in the MODY discovery cohort compared to the European control population within ExAC (odds ratio = 131, P = 1 × 10−4). We find similar results in non-Finnish European (n = 348, odds ratio = 43, P = 5 × 10−5) and Finnish (n = 80, odds ratio = 22, P = 1 × 10−6) replication cohorts. RFX6 heterozygotes have reduced penetrance of diabetes compared to common HNF1A and HNF4A-MODY mutations (27, 70 and 55% at 25 years of age, respectively). The hyperglycaemia results from beta-cell dysfunction and is associated with lower fasting and stimulated gastric inhibitory polypeptide (GIP) levels. Our study demonstrates that heterozygous RFX6 protein truncating variants are associated with MODY with reduced penetrance.

The mammalian lung develops through branching morphogenesis which is controlled by growth factors, hormones, and extracellular matrix proteins. We have evaluated the role of EGF-receptor signaling in lung morphogenesis by analyzing the developmental phenotype of lungs in mice with an inactivated the EGF-receptor gene both in vivo and in organ culture. Neonatal EGF-receptor-deficient mice often show evidence of lung immaturity which can result in visible respiratory distress. The lungs of these mutant mice had impaired branching and deficient alveolization and septation, resulting in a 50% reduction in alveolar volume and, thus, a markedly reduced surface for gas exchange. The EGF-receptor inactivation also resulted in type II pneumocyte immaturity, which was apparent from their increased glycogen content and a reduced number of lamellar bodies. The defective branching was already evident at Day 12 of embryonic development. When explants of embryonic lungs from Day 12 embryos were cultured under defined conditions, the branching defect in EGF-receptor-deficient lungs was even more pronounced, with only half as many terminal buds as normal lungs. EGF treatment stimulated the expression of surfactant protein C and thyroid transcription factor-1 in cultured normal lungs, but not in EGF-receptor-deficient lungs, suggesting that EGF-receptor signaling regulates the expression of these marker genes during type II pneumocyte maturation. Taken together, our data indicate that signal transduction through the EGF receptor plays a major role in lung development and that its inactivation leads to a respiratory distress-like syndrome.

BackgroundViral infections may trigger type 1 diabetes (T1D), and recent reports suggest an increased incidence of paediatric T1D and/or diabetic ketoacidosis (DKA) during the COVID-19 pandemic.ObjectiveTo study whether the number of children admitted to the paediatric intensive care unit (PICU) for DKA due to new-onset T1D increased during the COVID-19 pandemic, and whether SARS-CoV-2 infection plays a role.MethodsThis retrospective cohort study comprises two datasets: (1) children admitted to PICU due to new-onset T1D and (2) children diagnosed with new-onset T1D and registered to the Finnish Pediatric Diabetes Registry in the Helsinki University Hospital from 1 April to 31 October in 2016–2020. We compared the incidence, number and characteristics of children with newly diagnosed T1D between the prepandemic and pandemic periods.ResultsThe number of children admitted to PICU due to new-onset T1D increased from an average of 6.25 admissions in 2016–2019 to 20 admissions in 2020 (incidence rate ratio [IRR] 3.24 [95% CI 1.80 to 5.83]; p=0.0001). On average, 57.75 children were registered to the FPDR in 2016–2019, as compared with 84 in 2020 (IRR 1.45; 95% CI 1.13 to 1.86; p=0.004). 33 of the children diagnosed in 2020 were analysed for SARS-CoV-2 antibodies, and all were negative.ConclusionsMore children with T1D had severe DKA at diagnosis during the pandemic. This was not a consequence of SARS-CoV-2 infection. Instead, it probably stems from delays in diagnosis following changes in parental behaviour and healthcare accessibility.