The role of Wt1 in regulating mesenchyme in cancer, development, and tissue homeostasis

From both the fundamental and clinical perspectives, there is growing interest in mesenchymal cells and the mechanisms that regulate the two-way switch between mesenchymal and epithelial states. Here, we review recent findings showing that the Wilms' tumor gene (Wt1) is a key regulator of mesenchyme maintenance and the mesenchyme to epithelial balance in the development of certain mesodermal organs. We summarize recent experiments demonstrating, unexpectedly, that Wt1 is also essential for the integrity or function of multiple adult tissues, mainly, we argue, through regulating mesenchymal cells. We also discuss growing evidence that implicates Wt1 in tissue repair and regeneration. Drawing on these findings, we highlight the similarities between Wt1-expressing cells in different tissues. We believe that future studies aimed at elucidating the mechanisms underlying the functions of Wt1 in adult cells will reveal key cell types, pathways, and molecules regulating adult tissue homeostasis and repair.     

Increased serum IGF-1 levels protect the musculoskeletal system but are associated with elevated oxidative stress markers and increased mortality independent of tissue igf1 gene expression

Although the literature suggests a protective (anabolic) effect of insulin-like growth factor-1 (IGF-1) on the musculoskeletal system during growth and aging, there is evidence that reductions in IGF-1 signaling are advantageous for promoting an increase in life span through reduction in oxidative stress-induced tissue damage. To better understand this paradox, we utilized the hepatocyte-specific IGF-1 transgenic (HIT) mice, which exhibit 3-fold increases in serum IGF-1, with normal IGF-1 expression in other tissues, and mice with an IGF-1 null background that exclusively express IGF-1 in the liver, which thereby deliver IGF-1 by the endocrine route only (KO-HIT mice). We found that in the total absence of tissue igf1 gene expression (KO-HIT), increases in serum IGF-1 levels were associated with increased levels of lipid peroxidation products in serum and increased mortality rate at 18 months of age in both genders. Surprisingly, however, we found that in female mice, tissue IGF-1 plays an important role in preserving trabecular bone architecture as KO-HIT mice show bone loss in the femoral distal metaphysis. Additionally, in male KO-HIT mice, increases in serum IGF-1 levels were insufficient to protect against age-related muscle loss.     

An experimental model of partial insulin-like growth factor-1 deficiency in mice

Insulin-like growth factor-1 (IGF-1) is responsible for many systemic growth hormone (GH) functions although it has an extensive number of inherent activities (anabolic, cytoprotective, and anti-inflammatory). The potential options for IGF-1 therapy arise as a promising strategy in a wide list of human diseases. However, deeper studies are needed from a suitable animal model. All human conditions of IGF-1 deficiency consist in partially decreased IGF-1 levels since total absence of this hormone is hardly compatible with life. The aim of this work was to confirm that heterozygous Igf-1 ^+/? mice (Hz) may be considered as an appropriate animal model to study conditions of IGF-1 deficiency, focusing on early ages. Heterozygous Igf-1 ^+/? mice were compared to homozygous Igf-1 ^+/+ by assessing gene expression by quantitative PCR, serum circulating levels by ELISA, and tissue staining. Compared to controls, Hz mice (25 days old) showed a partial but significant reduction of IGF-1 circulating levels, correlating with a reduced body weight and diminished serum IGFBP-3 levels. Hz mice presented a significant decrease of IGF-1 gene expression in related organs (liver, bone, testicles, and brain) while IGF-1 receptor showed a normal expression. However, gene expression of growth hormone receptor (GHR) was increased in the liver but reduced in the bone, testicles, and brain. In addition, a significant reduction of cortical bone thickness and histopathological alterations in the testicles were found in Hz mice when compared to controls. Finally, the lifelong evolution of IGF-1 serum levels showed significant differences throughout life until aging in mice. Results in this paper provide evidence for considering heterozygous mice as a suitable experimental model, from early stages, to get more insight into the mechanisms of the beneficial actions induced by IGF-1 replacement therapy.     

Reduced levels of insulin-like growth factor-1 receptor (IGF-1R) suppress cellular signaling in experimental autoimmune sialadenitis (EAS).

The nonobese diabetic mouse (NOD) develops destruction and functional impairment of salivary and lachrymal glands, experimental autoimmune sialadenitis (EAS), resembling and representing a model for Sjogren's syndrome (SS). To investigate the mechanisms of tissue destruction in EAS, we analyzed a cell survival promoter insulin-like growth factor-1 receptor (IGF-1R) in the submandibular glands of NOD mice with this disease. We also evaluated the expression of a downstream effector of IGF-1R, BAD. Receptor-binding autoradiography revealed that the IGF-1R levels in submandibular glands from young NOD mice were lower than those in adult NOD mice. Immunofluorescence staining demonstrated that BAD expression in the epithelial cells of the submandibular gland was consistently enhanced throughout the course of EAS in NOD mice. These findings suggest that a reduction in the levels of IGF-1R induces a defective glandular homeostasis in the submandibular gland epithelial cells and triggers EAS.     

Matrix IGF-1 maintains bone mass by activation of mTOR in mesenchymal stem cells

Insulin-like growth factor 1 (IGF-1), the most abundant growth factor in the bone matrix, maintains bone mass in adulthood. We now report that IGF-1 released from the bone matrix during bone remodeling stimulates osteoblastic differentiation of recruited mesenchymal stem cells (MSCs) by activation of mammalian target of rapamycin (mTOR), thus maintaining proper bone microarchitecture and mass. Mice with knockout of the IGF-1 receptor (Igf1r) in their pre-osteoblastic cells showed lower bone mass and mineral deposition rates than wild-type mice. Further, MSCs from Igf1rflox/flox mice with Igf1r deleted by a Cre adenovirus in vitro, although recruited to the bone surface after implantation, were unable to differentiate into osteoblasts. We also found that the concentrations of IGF-1 in the bone matrix and marrow of aged rats were lower than in those of young rats and directly correlated with the age-related decrease in bone mass. Likewise, in age-related osteoporosis in humans, we found that bone marrow IGF-1 concentrations were 40% lower in individuals with osteoporosis than in individuals without osteoporosis. Notably, injection of IGF-1 plus IGF binding protein 3 (IGFBP3), but not injection of IGF-1 alone, increased the concentration of IGF-1 in the bone matrix and stimulated new bone formation in aged rats. Together, these results provide mechanistic insight into how IGF-1 maintains adult bone mass, while also providing a further rationale for its therapeutic targeting to treat age-related osteoporosis.     

Conditional knockout of mouse insulin-like growth factor-1 gene using the Cre/loxP system

Insulin-like growth factor-1 (IGF-1) is an essential growth factor for normal intrauterine development and postnatal growth. Mice with a complete deficiency of IGF-1 (IGF-1-null mice), created by homologous recombination, were found to exhibit postnatal lethality, growth retardation, infertility, and profound defects in the development of major organ systems. Furthermore, IGF-1-null mice were resistant to growth hormone (GH) treatment in peri-pubertal somatic growth. Using the Cre/loxP-induced conditional knockout system, we generated a mouse that lacks IGF-1 specifically in the liver, the primary site of IGF-1 production. Interestingly, although circulating and serum levels of IGF-1 were decreased by approximately 75% in these mice, they exhibited no defect in growth or development. When administered exogenously, GH stimulated IGF-1 production in several extra-hepatic tissues as well as body growth. The "Somatomedin hypothesis" originally proposed that circulating IGF-1 acting in various tissues mediate the effects of GH. These striking in vivo results, obtained using homologous recombination technology, call for a major modification of the Somatomedin hypothesis. These gene targeting studies confirm that IGF-1 is essential for GH-stimulated postnatal body growth. However, liver-derived (endocrine) IGF-1 is not essential for normal postnatal growth, though it does exert a negative feedback on GH secretion. Instead, local production of IGF-1, acting in a paracrine/autocrine fashion, appears to mediate GH-induced somatic growth. This review will discuss the effects of tissue-specific IGF-1 gene deficiency created by the Cre/loxP system versus the conventional IGF-1 knockout.     

Fas death receptor signaling represses monocyte numbers and macrophage activation in vivo

Over 1 billion monocytes are produced daily, with a small percentage differentiating into macrophages, suggesting that excess monocytes are deleted through a tightly regulated process. Although the in vivo mechanism governing monocyte/macrophage homeostasis is unknown, deletion of monocytes in culture is mediated by the Fas death pathway and is blocked by M-CSF. To determine the in vivo significance of Fas in monocyte development, mice lacking Fas (lpr/lpr) and mice deficient in Fas and M-CSF were examined. Compared with congenic control C57BL/6 (B6) mice, lpr/lpr mice displayed increased numbers of circulating monocytes. The lack of Fas in M-CSF-deficient mice resulted in an enhanced percentage, but not total numbers, of monocytes. Fas deficiency led to an increase in myeloid bone marrow progenitor potential only in M-CSF-intact mice. Although lpr/lpr and B6 mice had similar numbers of tissue macrophages, the loss of Fas in M-CSF-deficient mice was sufficient to increase the number of macrophages in a subset of tissues. Additionally, after stimulation with thioglycolate, lpr/lpr and B6 mice showed equivalent numbers of peritoneal macrophages. However, Fas-deficient peritoneal macrophages displayed a marked increase in spontaneous and LPS-induced proinflammatory molecule production. Moreover, Fas-deficient mice showed enhanced systemic inflammatory arthritis associated with up-regulation of IL-1beta and CCL2 secretion, elevated numbers of inflammatory monocytes, and increased numbers of tissue macrophages. Collectively, these data suggest that Fas may be required for maintaining circulating monocytes and for suppressing macrophage activation and recruitment that are stimulus dependent.     

Deletion of the developmentally essential gene ATR in adult mice leads to age-related phenotypes and stem cell loss

Developmental abnormalities, cancer, and premature aging each have been linked to defects in the DNA damage response (DDR). Mutations in the ATR checkpoint regulator cause developmental defects in mice (pregastrulation lethality) and humans (Seckel syndrome). Here we show that eliminating ATR in adult mice leads to defects in tissue homeostasis and the rapid appearance of age-related phenotypes, such as hair graying, alopecia, kyphosis, osteoporosis, thymic involution, fibrosis, and other abnormalities. Histological and genetic analyses indicate that ATR deletion causes acute cellular loss in tissues in which continuous cell proliferation is required for maintenance. Importantly, thymic involution, alopecia, and hair graying in ATR knockout mice were associated with dramatic reductions in tissue-specific stem and progenitor cells and exhaustion of tissue renewal and homeostatic capacity. In aggregate, these studies suggest that reduced regenerative capacity in adults via deletion of a developmentally essential DDR gene is sufficient to cause the premature appearance of age-related phenotypes.     

Generation of a conditional mouse model to target Acvr1b disruption in adult tissues

Alk4 is a type I receptor that belongs to the transforming growth factor‐beta (TGF‐β) family. It takes part in the signaling of TGF‐β ligands such as Activins, Gdfs, and Nodal that had been demonstrated to participate in numerous mechanisms ranging from early embryonic development to adult‐tissue homeostasis. Evidences indicate that Alk4 is a key regulator of many embryonic processes, but little is known about its signaling in adult tissues and in pathological conditions where Alk4 mutations had been reported. Conventional deletion of Alk4 gene (Acvr1b) results in early embryonic lethality prior gastrulation, which has precluded study of Alk4 functions in postnatal and adult mice. To circumvent this problem, we have generated a conditional Acvr1b floxed‐allele by flanking the fifth and sixth exons of the Acvr1b gene with loxP sites. Cre‐mediated deletion of the floxed allele generates a deleted allele, which behaves as an Acvr1b null allele leading to embryonic lethality in homozygous mutant animals. A tamoxifen‐inducible approach to target disruption of Acvr1b specifically in adult tissues was used and proved to be efficient for studying Alk4 functions in various organs. We report, therefore, a novel conditional model allowing investigation of biological role played by Alk4 in a variety of tissue‐specific contexts. genesis 51:120–127, 2013. © 2012 Wiley Periodicals, Inc.     

Dwarfism in mice lacking collagen-binding integrins α2β1 and α11β1 is caused by severely diminished IGF-1 levels

Mice with a combined deficiency in the α2β1 and α11β1 integrins lack the major receptors for collagen I. These mutants are born with inconspicuous differences in size but develop dwarfism within the first 4 weeks of life. Dwarfism correlates with shorter, less mineralized and functionally weaker bones that do not result from growth plate abnormalities or osteoblast dysfunction. Besides skeletal dwarfism, internal organs are correspondingly smaller, indicating proportional dwarfism and suggesting a systemic cause for the overall size reduction. In accordance with a critical role of insulin-like growth factor (IGF)-1 in growth control and bone mineralization, circulating IGF-1 levels in the sera of mice lacking either α2β1 or α11β1 or both integrins were sharply reduced by 39%, 64%, or 81% of normal levels, respectively. Low hepatic IGF-1 production resulted from diminished growth hormone-releasing hormone expression in the hypothalamus and, subsequently, reduced growth hormone expression in the pituitary glands of these mice. These findings point out a novel role of collagen-binding integrin receptors in the control of growth hormone/IGF-1-dependent biological activities. Thus, coupling hormone secretion to extracellular matrix signaling via integrins represents a novel concept in the control of endocrine homeostasis.     

Suitability of Nicotinic Acetylcholine Receptor α7 and Muscarinic Acetylcholine Receptor 3 Antibodies for Immune Detection: Evaluation in Murine Skin

Recent evidence reveals a crucial role for acetylcholine and its receptors in the regulation of inflammation, particularly of nicotinic acetylcholine receptor α7 (Chrna7) and muscarinic acetylcholine receptor 3 (Chrm3). Immunohistochemistry is a key tool for their cellular localization in functional tissues. We evaluated nine different commercially available antibodies on back skin tissue from wild-type (Wt) and gene-deficient (KO) mice. In the immunohistochemical analysis, we focused on key AChR-ligand sensitive skin cells (mast cells, nerve fibers and keratinocytes). All five antibodies tested for Chrm3 and the first three Chrna7 antibodies stained positive in both Wt and respective KO skin. With the 4th antibody (ab23832) nerve fibers were unlabeled in the KO mice. By western blot analysis, this antibody detected bands in both Wt and Chrna7 KO skin and brain. qRT-PCR revealed mRNA amplification with a primer set for the undeleted region in both Wt and KO mice, but none with a primer set for the deleted region in KO mice. By 2D electrophoresis, we found β-actin and β-enolase cross reactivity, which was confirmed by double immunolabeling. In view of the present results, the tested antibodies are not suitable for immunolocalization in skin and suggest thorough control of antibody specificity is required if histomorphometry is intended.     

Conditional deletion of the Bcl-x gene from erythroid cells results in hemolytic anemia and profound splenomegaly

Bcl-x is a member of the Bcl2 family and has been suggested to be important for the survival and maturation of various cell types including the erythroid lineage. To define the consequences of Bcl-x loss in erythroid cells and other adult tissues, we have generated mice conditionally deficient in the Bcl-x gene using the Cre-loxP recombination system. The temporal and spatial excision of the floxed Bcl-x locus was achieved by expressing the Cre recombinase gene under control of the MMTV-LTR. By the age of five weeks, Bcl-x conditional mutant mice exhibited hyperproliferation of megakaryocytes and a decline in the number of circulating platelets. Three-month-old animals suffered from severe hemolytic anemia, hyperplasia of immature erythroid cells and profound enlargement of the spleen. We demonstrate that Bcl-x is only required for the survival of erythroid cells at the end of maturation, which includes enucleated reticulocytes in circulation. The extensive proliferation of immature erythroid cells in the spleen and bone marrow might be the result of a fast turnover of late red blood cell precursors and accelerated erythropoiesis in response to tissue hypoxia. The increase in cell death of late erythroid cells is independent from the proapoptotic factor Bax, as demonstrated in conditional double mutant mice for Bcl-x and Bax. Mice conditionally deficient in Bcl-x permitted us for the first time to study the effects of Bcl-x deficiency on cell proliferation, maturation and survival under physiological conditions in an adult animal.     

Micro RNAs: tiny sequences with enormous potential

The liver plays a central role in glucose homeostasis in the whole-body by responding to environmental factors including nutrients, hormones, and oxygen. In conditions of metabolic overload such as diabetes mellitus and obesity, coordinated regulation between oxygen supply and consumption has been reported to be disrupted and subsequently cause tissue hypoxia, although pathological significance of the disease-related hypoxia remains elusive. To investigate the role of tissue hypoxia in the liver on systemic glucose homeostasis, mice lacking HIF-1α gene, a critical component of a master regulator of hypoxic response, in hepatocytes were exposed to high fat/sucrose diet (HFSD). Exposure to HFSD for 5 weeks elicited liver hypoxia with a transient increase in HIF-1α protein expression in the liver of control mice. Glucose disposal was marginally impaired in control mice when challenged oral glucose tolerance test, but such impairment was enhanced in the mutant mice. This alteration was accompanied by a complete inhibition of glucokinase induction with a significant reduction of hepatic glucose uptake. Mice fed HFSD for 20 weeks exhibited fasting hyperglycemia and glucose intolerance, whereas these metabolic phenotypes deteriorated considerably with severe insulin resistance in skeletal muscles and adipose tissues in the mutant mice. These findings suggest that HIF-1 in hepatocytes plays protective roles against the progression of diabetes mellitus.     

Alk3 mediated Bmp signaling controls the contribution of epicardially derived cells to the tissues of the atrioventricular junction

Recent studies using mouse models for cell fate tracing of epicardial derived cells (EPDCs) have demonstrated that at the atrioventricular (AV) junction EPDCs contribute to the mesenchyme of the AV sulcus, the annulus fibrosus, and the parietal leaflets of the AV valves. There is little insight, however, into the mechanisms that govern the contribution of EPDCs to these tissues. While it has been demonstrated that bone morphogenetic protein (Bmp) signaling is required for AV cushion formation, its role in regulating EPDC contribution to the AV junction remains unexplored. To determine the role of Bmp signaling in the contribution of EPDCs to the AV junction, the Bmp receptor activin-like kinase 3 (Alk3; or Bmpr1a) was conditionally deleted in the epicardium and EPDCs using the mWt1/IRES/GFP-Cre (Wt1^Cre) mouse. Embryonic Wt1^Cre;Alk3^fl/fl specimens showed a significantly smaller AV sulcus and a severely underdeveloped annulus fibrosus. Electrophysiological analysis of adult Wt1^Cre;Alk3^fl/fl mice showed, unexpectedly, no ventricular pre-excitation. Cell fate tracing revealed a significant decrease in the number of EPDCs within the parietal leaflets of the AV valves. Postnatal Wt1^Cre;Alk3^fl/fl specimens showed myxomatous changes in the leaflets of the mitral valve. Together these observations indicate that Alk3 mediated Bmp signaling is important in the cascade of events that regulate the contribution of EPDCs to the AV sulcus, annulus fibrosus, and the parietal leaflets of the AV valves. Furthermore, this study shows that EPDCs do not only play a critical role in early developmental events at the AV junction, but that they also are important in the normal maturation of the AV valves.     

Adiponectin plays an important role in efficient energy usage under energy shortage

Adiponectin is an adipose tissue-specific secretory protein known to be an insulin-sensitizing protein. In this study, we generated adiponectin sense and antisense transgenic (Tg) mice to investigate whether adiponectin plays a role in the regulation of energy homeostasis during the growth stage. Spontaneous motor activity of antisense Tg mice were markedly reduced during fasting, particularly in young female mice, compared with wild type (Wt) and sense Tg mice. Furthermore, both body weight and adipose tissue mass of the antisense female Tg mice drastically reduced during fasting. To examine the relationship between the collapse of abdominal white adipose tissue (WAT) and serum adiponectin level, we measured the expression of genes related to energy expenditure, such as uncoupling protein (UCP). Notably, the mRNA of UCP1 in the WAT of antisense Tg female mice was markedly less than that of Wt mice and the UCP1 mRNA was strongly increased during fasting. These findings suggest that the serum adiponectin is important to maintaining energy homeostasis under energy shortage conditions, such as over female pubertal development.