Smad1 and Its Target Gene Wif1 Coordinate BMP and Wnt Signaling
Total Page：16
File Type：pdf, Size：1020Kb
RESEARCH ARTICLE 925 Development 138, 925-935 (2011) doi:10.1242/dev.062687 © 2011. Published by The Company of Biologists Ltd Smad1 and its target gene Wif1 coordinate BMP and Wnt signaling activities to regulate fetal lung development Bing Xu1,*, Cheng Chen1,2,*, Hui Chen1, Song-Guo Zheng3, Pablo Bringas, Jr4, Min Xu5, Xianghong Zhou5, Di Chen6, Lieve Umans7, An Zwijsen7 and Wei Shi1,4,† SUMMARY Bone morphogenetic protein 4 (Bmp4) is essential for lung development. To define the intracellular signaling mechanisms by which Bmp4 regulates lung development, BMP-specific Smad1 or Smad5 was selectively knocked out in fetal mouse lung epithelial cells. Abrogation of lung epithelial-specific Smad1, but not Smad5, resulted in retardation of lung branching morphogenesis and reduced sacculation, accompanied by altered distal lung epithelial cell proliferation and differentiation and, consequently, severe neonatal respiratory failure. By combining cDNA microarray with ChIP-chip analyses, Wnt inhibitory factor 1 (Wif1) was identified as a novel target gene of Smad1 in the developing mouse lung epithelial cells. Loss of Smad1 transcriptional activation of Wif1 was associated with reduced Wif1 expression and increased Wnt/-catenin signaling activity in lung epithelia, resulting in specific fetal lung abnormalities. This suggests a novel regulatory loop of Bmp4-Smad1-Wif1-Wnt/-catenin in coordinating BMP and Wnt pathways to control fetal lung development. KEY WORDS: Lung morphogenesis, Bone morphogenetic protein, Smad1, Wif1, Mouse INTRODUCTION lung developmental processes are regulated by coordinated Disruption of normal lung development can result in a variety of signaling networks, including bone morphogenetic proteins neonatal respiratory problems (Warburton et al., 2006b), although (BMPs) and Wnts (Cardoso, 2001; Warburton et al., 2006a). the molecular mechanism of lung development and its relationship Among the many BMP ligands, Bmp4 has been identified as a to neonatal pulmonary diseases remains incompletely understood. major ligand involved in lung developmental regulation (Eblaghie Lung development is composed of early branching morphogenesis et al., 2006). However, the specific intracellular signaling pathway and late alveogenesis processes. Through branching that mediates BMP regulatory effects during lung development has morphogenesis, the lung epithelial buds undergo reiterated not been defined. Studies in vitro have demonstrated that BMP outgrowth, elongation and subdivision to form complicated airway ligands bind to heteromeric BMP receptor complexes and activate structures, accompanied by vasculogenesis (Hogan, 1999; Metzger the receptor serine/threonine kinases, which subsequently et al., 2008). Then, terminal air sacs and alveoli are formed to phosphorylate cytoplasmic Smad1, Smad5 and Smad8 (also known generate a large gas-exchange surface. At the same time, distal lung as Smad9 – Mouse Genome Informatics). These phosphorylated epithelial cells differentiate into type I and type II alveolar Smads then dissociate from the receptors, form complexes with epithelial cells (AECI and AECII). Cuboidal AECII undergo Smad4, translocate into the nucleus and bind to BMP-responsive marked ultrastructural and biochemical changes, including elements in their target genes to modulate gene expression (Shi and depletion of glycogen, increased synthesis of surfactant proteins Massague, 2003). In addition, Smad-independent pathways have and lipids, increased numbers of lamellar bodies and secretion of also been reported to transduce BMP signals (Derynck and Zhang, surfactant proteins into the air space, which reduces surface tension 2003). Previous studies by us and other groups have found that at the air-liquid interface, while flat AECI cover the majority of the Alk3 [also known as BMP type IA receptor (Bmpr1a)], rather than airspace surface and provide the capacity for fluid transportation Alk2 (Acvr1) and Alk6 (Bmpr1b), plays an essential role in across the respiratory membrane (Cardoso and Whitsett, 2008; mediating BMP regulatory signals during prenatal lung Whitsett and Weaver, 2002; Williams, 2003). These complicated development in mice (Eblaghie et al., 2006; Sun et al., 2008). Abrogation of Alk3 in developing mouse lung epithelial cells causes immediate neonatal respiratory distress and lethality due to 1Developmental Biology and Regenerative Medicine Program, Childrens Hospital Los abnormal fetal lung formation and postnatal lung atelectasis. Angeles, Keck School of Medicine, University of Southern California, Los Angeles, In order to further define the intracellular BMP signaling CA 90027, USA. 2Department of Developmental Biology, China Medical University, Shenyang, Liaoning 110001, The People’s Republic of China. 3Department of mechanisms that regulate lung development, the role of BMP- Medicine, Keck School of Medicine, University of Southern California, CA 90033, specific downstream Smads must be addressed. As reported by 4 USA. Center for Craniofacial Molecular Biology, School of Dentistry, University of other groups, blockade of Smad8 function by a conventional Southern California, Los Angeles, CA 90033, USA. 5Molecular and Computational Biology, Department of Biological Sciences, University of Southern California, Los knockout approach in mice has no significant impact on fetal lung Angeles, CA 90089, USA. 6Department of Orthopaedics, Center for Musculoskeletal formation, although pulmonary arterial medial thickening and Research, University of Rochester School of Medicine, Rochester, NY 14642, USA. pulmonary adenoma are observed in the adult Smad8 knockout 7VIB Department of Molecular and Developmental Genetics and Center for Human Genetics, KULeuven, 3000 Leuven, Belgium. lung (Arnold et al., 2006; Huang et al., 2009). Therefore, we focused on Smad1 and Smad5 in lung development in vivo. We *These authors contributed equally to this work employed a loxP/Cre genetic approach to generate lung epithelial †Author for correspondence ([email protected]) cell-specific conditional gene knockout mice, as conventional Accepted 14 December 2010 deletion of the Smad1 or Smad5 gene results in early embryonic DEVELOPMENT 926 RESEARCH ARTICLE Development 138 (5) lethality before lung organogenesis (Chang et al., 1999; Lechleider Western blot and densitometric analysis et al., 2001; Tremblay et al., 2001; Yang et al., 1999). Furthermore, Protein detection in lung tissue by western blot has been described we also explored specific Smad1-modulated target genes in previously (Sun et al., 2006). Proteins were detected by specific antibodies developing fetal mouse lung at the transcriptional level. One of against Smad1 (Millipore), Wif1 (goat polyclonal, Santa Cruz), active - these is Wnt inhibitory factor 1 (Wif1), which encodes a factor that catenin (8E7, mouse monoclonal, Millipore), phosphorylated Lrp6 [Ab1490, kindly provided by Dr Xi He at Harvard Medical School (He et acts as an antagonist for Wnt ligand and negatively regulates Wnt al., 2004)], -actin (Santa Cruz) and Gapdh (6C5, mouse monoclonal, signaling activity (Hsieh et al., 1999). Reduction of Wif1 gene Fitzgerald). The intensities of the protein bands were quantified using expression and the subsequent increase in -catenin activation Image J software and normalized to the protein loading control (Gapdh). result in specific distal lung abnormalities in Smad1 conditional knockout fetal lung. Based on the data presented here, we define RNA isolation and real-time PCR analysis for the first time the intracellular BMP signaling pathway that is Total RNA was isolated from lung tissues using the RNeasy kit (Qiagen) involved in regulating fetal lung development, and then propose a following the manufacturer’s protocol. Real-time PCR reactions were performed as described previously (Chen et al., 2008a). Primer sequences novel mechanism for signal crosstalk between the BMP and Wnt are described in our previous publication (Sun et al., 2008). pathways in controlling lung organogenesis and homeostasis. cDNA microarray analysis MATERIALS AND METHODS Total RNA (20 g) isolated from E18.5 wild-type and Smad1 Ep-CKO Mouse strains and breeding lungs was used to prepare biotinylated cRNA according to the protocol Floxed Smad1 mice were generated in the laboratory of Dr Anita B. described in the Affymetrix expression analysis technical manual. Samples Roberts (Huang et al., 2002), in which the Smad1 gene locus was targeted from three pairs of Smad1 Ep-CKO mice and wild-type controls were used by loxP-PGKneo-loxP-exon2-loxP. We then removed the PGKneo cassette to hybridize with GeneChip Mouse Genome 430 2.0 arrays (Affymetrix). by crossing the floxed Smad1 heterozygous mice with EIIa-Cre mice (Xu Images were acquired using a GeneChip Scanner 3000. Data were et al., 2001) to generate the Smad1fx allele (loxP-exon2-loxP) in order to analyzed using an RMA method to perform background adjustment and avoid non-specific genetic interference caused by PGKneo cassette quantitative normalization (Bolstad et al., 2003). The expression ratio insertion. The Smad1 heterozygous mouse line (Smad1+/–) was also derived between knockout and wild-type samples was computed. Probes that did from the above crossbreeding. Floxed Smad5 mice were as published not have detectable signals in both samples were discarded. Three pairs of previously (Umans et al., 2003). Smad5+/– mice were derived by crossing experiments were then combined, and genes that were all upregulated or Smad5+/fx with X-linked CMV-Cre mice