Nora Heisterkamp

Transforming growth factor‐β3 regulates transdifferentiation of medial edge epithelium during palatal fusion and associated degradation of the basement membrane

Studies on transforming growth factor β3 (TGF‐β3) deficient mice have shown that TGF‐β3 plays a critical role in palatogenesis. These null mutant mice have clefting of the secondary palate, caused by a defect in the process of fusion of the palatal shelves. A critical step in mammalian palatal fusion is removal of the medial edge epithelial cells from the midline seam and formation of continuous mesenchyme. To determine in more detail the role of TGF‐β3 in palatogenesis, we cultured TGF‐β3 null mutant and wild‐type control palatal shelves in an organ culture system. The fate of the medial edge epithelial cells was studied in vitro using vital cell labeling and immunohistochemical techniques. Despite clear adherence, the null mutant palatal shelves did not fuse in vitro, but instead the medial edge epithelial cells survived at the midline position, and the basement membrane was resistant towards degradation. Supplementation of the culture medium with the mature form of TGF‐β3 was able to fully correct the defective fusion in the null mutant specimens. Our results demonstrate that the reason for the defective palatal fusion in TGF‐β3 (−/−) samples is not impaired adhesion. Our data define a specific role for TGF‐β3 in the events that control transdifferentiation of the medial edge epithelial cells including degradation of the underlying basement membrane. Dev. Dyn. 209:255–260, 1997.

TGFβ signaling plays a critical role in promoting alternative macrophage activation

BackgroundUpon stimulation with different cytokines, macrophages can undergo classical or alternative activation to become M1 or M2 macrophages. Alternatively activated (or M2) macrophages are defined by their expression of specific gene products and play an important role in containing inflammation, removing apoptotic cells and repairing tissue damage. Whereas it is well-established that IL-4 can drive alternative activation, if lack of TGFβ signaling at physiological levels affects M2 polarization has not been addressed.ResultsVav1-Cre x TβRIIfx/fx mice, lacking TβRII function in hematopoietic cells, exhibited uncontrolled pulmonary inflammation and developed a lethal autoimmune syndrome at young age. This was accompanied by significantly increased numbers of splenic neutrophils and T cells as well as elevated hepatic macrophage infiltration and bone marrow monocyte counts. TβRII-/- CD4+ and CD8+ T-cells in the lymph nodes and spleen expressed increased cell surface CD44, and CD69 was also higher on CD4+ lymph node T-cells. Loss of TβRII in bone marrow-derived macrophages (BMDMs) did not affect the ability of these cells to perform efferocytosis. However, these cells were defective in basal and IL-4-induced arg1 mRNA and Arginase-1 protein production. Moreover, the transcription of genes that are typically upregulated in M2-polarized macrophages, such as ym1, mcr2 and mgl2, was also decreased in peritoneal macrophages and IL-4-stimulated TβRII-/- BMDMs. We found that cell surface and mRNA expression of Galectin-3, which also regulates M2 macrophage polarization, was lower in TβRII-/- BMDMs. Very interestingly, the impaired ability of these null mutant BMDMs to differentiate into IL-4 polarized macrophages was Stat6- and Smad3-independent, but correlated with reduced levels of phospho-Akt and β-catenin.ConclusionsOur results establish a novel biological role for TGFβ signaling in controlling expression of genes characteristic for alternatively activated macrophages. We speculate that lack of TβRII signaling reduces the anti-inflammatory M2 phenotype of macrophages because of reduced expression of these products. This would cause defects in the ability of the M2 macrophages to negatively regulate other immune cells such as T-cells in the lung, possibly explaining the systemic inflammation observed in Vav1-Cre x TβRIIfx/fx mice.

Enzyme replacement therapy in a mouse model of aspartylglycosaminuria

Aspartylglycosaminuria (AGU), the most common lysosomal disorder of glycoprotein degradation, is caused by deficient activity of glycosylasparaginase (AGA). AGA‐deficient mice share most of the clinical, biochemical and histopathologic characteristics of human AGU disease. In the current study, recombinant human AGA administered i.v. to adult AGU mice disappeared from the systemic circulation of the animals in two phases predominantly into non‐neuronal tissues, which were rapidly cleared from storage compound aspartylglucosamine. Even a single AGA injection reduced the amount of aspartylglucosamine in the liver and spleen of AGU mice by 90% and 80%, respectively. Quantitative biochemical analyses along with histological and immunohistochemical studies demonstrated that the pathophysiologic characteristics of AGU were effectively corrected in non‐neuronal tissues of AGU mice during 2 wk of AGA therapy. At the same time, AGA activity increased to 10% of that in normal brain tissue and the accumulation of aspartylglucosamine was reduced by 20% in total brain of the treated animals. Immunohistochemical studies suggested that the corrective enzyme was widely distributed within the brain tissue. These findings suggest that AGU may be correctable by enzyme therapy.—Dunder, U., Kaartinen, V., Valtonen, P., Väänänen, E., Kosma, V.‐M., Heisterkamp, N., Groffen, J., Mononen, I. Enzyme replacement therapy in a mouse model of aspartylglycosaminuria. FASEB J. 14, 361–367 (2000)

Nuclear and cytoplasmic location of the FER tyrosine kinase.

The location of the FER protein within the cell was investigated by using subcellular fractionation and immunofluorescence. FER was found in the cytoplasm and in the nucleus, where it was associated with the chromatin fraction. Its ubiquitous expression and its subcellular location indicate that it may be involved in key regulatory processes.

Localization of a gamma-glutamyl-transferase-related gene family on chromosome 22

A gene family encompassing a minimum of four genes or pseudogenes for gamma-glutamyl transferase (GGT; EC 2.3.2.2) is present on chromosome 22q11. We have previously isolated a cDNA related to GGT but clearly not belonging to its gene family. The chromosomal location of this related gene, GGTLA1, has been determined by both isotopic and fluorescence in situ hybridization to metaphase cells and by Southern blot analysis of somatic cell hybrid DNAs. We show that GGTLA1 is part of a distinct gene family, which has at least four members (GGTLA1, GGTLA2, GGTLA3, GGTLA4). At least two loci are located on chromosome 22 within band q11 and proximal to the chronic myelogenous leukemia (CML) breakpoint in BCR (breakpoint cluster region gene). At least one other member is located more distally between the breakpoints found in Ewings sarcoma and CML. Some of the GGT and GGTLA family members are located on NotI restriction enzyme fragments of a similar size. Combined results indicate that a segment of human chromosome 22q11 has undergone largescale amplification events relatively recently in evolution.

The human tyrosine kinase gene (FER) maps to chromosome 5 and is deleted in myeloid leukemias with a del(5q)

A novel member of the SRC tyrosine kinase gene family was recently isolated and characterized (Hao et al., 1989). This FES/FPS-related gene, named FER, lacks the transmembrane and extracellular domains which characterize tyrosine kinases with receptor function. Expression of FER in a wide range of cell types indicates a general role in intracellular signalling or differentiation processes. We have now mapped FER to chromosome 5q14----q23 using in situ hybridization techniques and suggest a more precise location within bands 5q21----q22. This region lies adjacent to a complex domain of growth factors and receptors, many involved in regulation of haematopoiesis. FER maps within a critical segment frequently deleted from chromosome 5 in patients with acute myeloid leukemia or myelodysplastic syndromes and was shown to be deleted in two such patients. It also maps close to the familial polyposis coli locus at 5q22.

Recombinant glycosylasparaginase and in vitro correction of aspartylglycosaminuria.

Aspartylglycosaminuria (AGU) is the most common disorder of glycoprotein degradation. AGU patients are deficient in glycosylasparaginase (GA), which results in accumulation of aspartylglucosamine in body fluids and tissues. Human glycosylasparaginase was stably overexpressed in NIH‐3T3 mouse fibroblasts, in which the unusual posttranslational processing and maturation of the enzyme occurred in a high degree. The recombinant enzyme was isolated as two isoforms, which were both phosphorylated, and actively transported into AGU fibroblasts and lymphoblasts through mannose‐6‐phosphate receptor‐mediated endocytosis. The rate of uptake into fibroblasts was half‐maximal when the concentration of GA in the medium was 5 × 10–8 M. Immunofluorescence microscopy suggested compartmentalization of the recombinant enzyme in the lysosomes. Supplementation of culture medium with either isoform cleared AGU lymphoblasts of stored aspartylglucosamine when glycosylasparaginase activity in the cells reached 3–4% of that in normal lymphoblasts. A relatively small amount of recombinant GA in the culture medium was sufficient to reverse pathology in the target cells, indicating high corrective quality of the enzyme preparations. The combined evidence indicates that enzyme replacement therapy with the present recombinant glycosylasparaginase might reverse pathology at least in somatic cells of AGU patients.—Mononen, I., Heisterkamp, N., Dunder, U., Romppanen, E‐L., Noronkoski, T., Kuronen, I., Groffen, J. Recombinant glycosylasparaginase and in vitro correction of aspartylglycosaminuria. FASEB J. 9, 428–433 (1995)

Chromosomal localization of the human glycoasparaginase gene to 4q32–q33

SummaryGlycoasparaginase cleaves the N-glycosidic linkage between asparagine and N-acetylglucosamine in the degradation of glycoproteins. In humans, a deficient activity of glycoasparaginase results in accumulation of glycoasparagines, causing the lysosomal storage disease aspartylglycosaminuria. Recombinant plasmid containing the cDNA insert encoding human glycoasparaginase was used to localize the enzyme to chromosome 4q32–q33 by in situ hybridization to metaphase chromosomes prepared from normal human lymphocytes.

Ph-positive Leukemia: A Transgenic Mouse Model

The presence of the BCR/ABL chimeric gene is the hallmark of defined types of human leukemia. To increase our knowledge of the oncogenic processes and to develop a model for this type of leukemia we generated a BCR/ABL (P190) transgenic mouse line. Over 95% of mice of this line die of leukemia or leukemia/lymphoma within 35-200 days of age. Karyotypically visible genetic alterations were absent from the early stages of BCR/ABL generated leukemia. A high frequency of aneuploidy was found in advanced leukemia indicating a primary and pivotal role for BCR/ABL in leukemogenesis. Moreover, the data suggest that BCR/ABL has a destabilizing effect on the regulation of the cell cycle. BCR/ABL expression was also found in tissues other than hematopoietic cells. However, this did not result in the development of solid tumors, strongly suggesting that the oncogenicity of BCR/ABL is limited to the hematopoietic lineage.

Cell type–specific expression of adenomatous polyposis coli in lung development, injury, and repair

Adenomatous polyposis coli (Apc) is critical for Wnt signaling and cell migration. The current study examined Apc expression during lung development, injury, and repair. Apc was first detectable in smooth muscle layers in early lung morphogenesis, and was highly expressed in ciliated and neuroendocrine cells in the advanced stages. No Apc immunoreactivity was detected in Clara or basal cells, which function as stem/progenitor cell in adult lung. In ciliated cells, Apc is associated mainly with apical cytoplasmic domain. In response to naphthalene‐induced injury, Apcpositive cells underwent squamous metaplasia, accompanied by changes in Apc subcellular distribution. In conclusion, both spatial and temporal expression of Apc is dynamically regulated during lung development and injury repair. Differential expression of Apc in progenitor vs. nonprogenitor cells suggests a functional role in cell‐type specification. Subcellular localization changes of Apc in response to naphthalene injury suggest a role in cell shape and cell migration. Developmental Dynamics 239:2288–2297, 2010.