Mice deficient in Abl are osteoporotic and have defects in osteoblast maturation.

The c-Abl protein is a non-receptor tyrosine kinase involved in many aspects of mammalian development. c-Abl kinase is widely expressed, but high levels are found in hyaline cartilage in the adult, bone tissue in newborn mice, and osteoblasts and associated neovasculature at sites of endochondrial ossification in the fetus. Mice homozygous for mutations in the gene encoding c-Abl (AIM) display increased perinatal mortality, reduced fertility, foreshortened crania and defects in the maturation of B cells in bone marrow. Here we demonstrate that Abl-/- mice are also osteoporotic. The long bones of mutant mice contain thinner cortical bone and reduced trabecular bone volume. The osteoporotic phenotype is not due to accelerated bone turnover--both the number and activity of osteoclasts are similar to those of control littermates--but rather to dysfunctional osteoblasts. In addition, the rate of mineral apposition in the mutant animals is reduced. Osteoblasts from both stromal and calvarial explants showed delayed maturation in vitro as measured by expression of alkaline phosphatase (ALP), induction of mRNA encoding osteocalcin and mineral deposition.

Tyro-3 family receptors are essential regulators of mammalian spermatogenesis.

We have generated and analysed null mutations in the mouse genes encoding three structurally related receptors with tyrosine kinase activity: Tyro 3, Axl, and Mer. Mice lacking any single receptor, or any combination of two receptors, are viable and fertile, but male animals that lack all three receptors produce no mature sperm, owing to the progressive death of differentiating germ cells. This degenerative phenotype appears to result from a failure of the tropic support that is normally provided by Sertoli cells of the seminiferous tubules, whose function depends on testosterone and additional factors produced by Leydig cells. Tyro 3, Axl and Mer are all normally expressed by Sertoli cells during postnatal development, whereas their ligands, Gas6 and protein S, are produced by Leydig cells before sexual maturity, and by both Leydig and Sertoli cells thereafter. Here we show that the concerted activation of Tyro 3, Axl and Mer in Sertoli cells is critical to the role that these cells play as nurturers of developing germ cells. Additional observations indicate that these receptors may also be essential for the tropic maintenance of diverse cell types in the mature nervous, immune and reproductive systems.

Transforming activity of retroviral genomes encoding Gag-Axl fusion proteins.

Retroviral genomes encoding a portion of the Moloney murine leukemia virus Gag protein fused to portions of the murine axl cDNA were constructed so as to mimic naturally occurring transforming viruses. Virus MA1 retained 5 amino acids of the extracellular domain and the complete transmembrane and intracellular domains of Axl; virus MA2 retained only the intracellular Axl sequences beginning 33 amino acids downstream of the transmembrane region. Although both viruses could transform NIH 3T3 cells, they induced different morphological changes. MA1 transformants became elongated and assumed a cross-hatched pattern, while MA2 transformants were round and very refractile and grew to high density. Gag-Axl and Glyco-Gag-Axl proteins were detected in both types of transformed cells and were predominantly localized to the cytoplasmic compartment. When cell-free v-axl virus supernatants were introduced into wild-type BALB/c neonates, Rag-2-deficient mice, or c-myc transgenic mice, they did not cause tumors in a 3-month period. However, MA2-transformed NIH 3T3 cells, but not MA1 or control cells, could establish sarcomas by subcutaneous or intraperitoneal injection into BALB/c neonates. These results show that the transforming potential of the axl gene can be activated by truncation of the extracellular domain of the receptor and fusion of the remaining sequence to the gag gene.

Abnormal peripheral lymphocyte function in c-abl mutant mice.

The proto-oncogene c-abl encodes a tyrosine kinase that is hypothesized to function in proliferation-stimulatory signaling pathways. Previous work on mice homozygous for targeted mutations in the c-abl gene (ablml and abl2 mutant strains) has demonstrated multiple defects, including a susceptibility to infections that results in a high mortality rate after weaning. FACS analysis of the hemopoietic system of c-abl mutants demonstrated variable reductions in B and T lymphocytes in adult bone marrow, thymus, spleen, and peripheral blood. In addition, bone marrow from mutants showed a decreased ability to respond to interleukin-7. We further found that B cells from ablm1 mice had a reduced ability to respond to lipopolysaccharide (decreased to 10% of control response) that was dependent on the culture conditions and the tissue of origin of B cells. Peripheral blood from the mutants also had a reduced response to the T cell mitogen concanavalin A. Immune response in ablm1 mice as determined by the mixed lymphocyte response and the sheep red blood cell plaque-forming assay was grossly normal. These findings suggest that although specific signaling pathways in lymphocytes may involve c-Abl, the immune system can function in the absence of a normal c-abl gene product.

Transgenes encoding both type I and type IV c-abl proteins rescue the lethality of c-abl mutant mice.

Mice carrying homozygous mutations in the c-abl gene (abl-(m1) or abl2) exhibit severe, though variable phenotypes, including a high rate of postnatal mortality, runting, morphological abnormalities, a susceptibility to infections, and selected immune system defects. To further determine the role of the c-Abl protein in vivo, we have generated three lines of mice expressing c-abl transgenes. These minigenes encode the two major forms of the c-abl gene product (c-Abl types I and IV) and a kinase defective type IV c-Abl. The transgenic lines, in Abl-positive genetic backgrounds, were phenotypically almost indistinguishable from their non-transgene littermates and expressed the c-abl transgene in a variety of tissues at levels comparable to that of the endogenous c-abl gene. When the transgenes were introduced into a mutant c-abl strain by mating, the mutant c-abl phenotype was almost completely rescued by either of the c-abl type I or type IV transgenes, but not by the kinase-defective transgene. These findings suggest that either of the two alternatively spliced c-abl gene products can provide the in vivo functions of c-Abl, and that these functions are dependent on kinase activity.

Bone marrow B lymphocyte development in c-abl-deficient mice.

Mice homozygous for a mutation in the c-abl tyrosine kinase gene have multiple defects including high postnatal mortality, runting, morphological abnormalities, susceptibility to infections, and reductions in lymphocytes and their precursors. FACS analysis of bone marrow from mutant mice demonstrates variable reductions in pro-B and pre-B cells. While the numbers of cells in these populations are profoundly reduced in some mutants (16 and 1.2% of control pro-B and pre-B cells, respectively), normal levels are found in other individuals. In the affected mutants, some reductions are observed in many stages of B cell development. The response of B cell precursors to the cytokine interleukin-7 is variably affected while that of several other cytokines (stem cell factor, interleukin-3, GM-CSF, G-CSF, and erythropoietin) is normal in c-abl mutants. The population defects caused by the c-abl mutation can be recreated in normal mice by the transfer of adult bone marrow but, surprisingly, not fetal liver. These studies demonstrate that c-Abl signaling pathways may play a role in the earliest stages of B cell development in a developmental stage-specific manner. In spite of these variable abnormalities, however, the hemopoietic system of c-abl mutant animals is surprisingly intact.

Targeted disruption of the flk2/flt3 gene leads to deficiencies in primitive hematopoietic progenitors.

The flk2 receptor tyrosine kinase has been implicated in hematopoietic development. Mice deficient in flk2 were generated. Mutants developed into healthy adults with normal mature hematopoietic populations. However, they possessed specific deficiencies in primitive B lymphoid progenitors. Bone marrow transplantation experiments revealed a further deficiency in T cell and myeloid reconstitution by mutant stem cells. Mice deficient for both c-kit and flk2 exhibited a more severe phenotype characterized by large overall decreases in hematopoietic cell numbers, further reductions in the relative frequencies of lymphoid progenitors, and a postnatal lethality. Taken together, the data suggest that flk2 plays a role both in multipotent stem cells and in lymphoid differentiation.

A role for c-Abl in c-myc regulation.

c-Abl, a nonreceptor tyrosine kinase, appears to play a role in cell cycle progression, cell proliferation and differentiation. Mice homozygous for a mutation in c-abl (ablml), show pleiotropic abnormalities, including neonatal death, developmental defects, susceptibility to infection and dehydration (Schwartzberg et al., 1991). However, the exact substrates of c-Abl and the signal transduction pathways it might initiate are not known. We have examined how c-Abl affects c-myc expression by studying ablml mice. Quantitative riboprobe analyses demonstrated that in the heart, liver, thymus, brain, testes, intestines and lung, there were no differences in the steady-state level of c-myc RNA between the ablml mice and littermate controls. However, in adrenal glands, kidneys and splenic B cells, c-myc RNA levels were decreased approximately 50% compared to littermate controls. Induction of c-myc mRNA following activation of splenic B cells with LPS is also defective in ablml splenocytes. Finally, we show that c-Abl can directly transactivate c-myc transcription. These results suggest that c-Abl is involved in the normal transcription regulation of c-myc in selected tissues and that decreased c-myc RNA could be one cause of abnormalities in the ablml mice.

The abolition of collagen gene expression in SV40-transformed fibroblasts is associated with trans-acting factor switching.

The goal of this study was to determine whether alpha 2(1) procollagen gene expression is modulated by positive or negative trans-acting DNA-binding proteins. Previous studies have shown that a clone of SV40-transformed WI-38 fibroblasts (SVWI-38) does not produce any alpha 2(1) procollagen mRNA (Parker et al (1989), J. Biol Chem. 264, 7147-7152). In order to elucidate the mechanism(s) responsible for such inactivation, we have examined the activity of a transfected wild type COL1A2 promoter in SVWI-38 cells. A set of 5' promoter deletions was linked to the chloramphenicol acetyltransferase (CAT) gene and transfected into SVWI-38 and other cell lines expression type I collagen. The resulting CAT assays confirmed the importance of several upstream regions for promoter activity and documented the decreased transcriptional activity from an exogenous COL1A2 promoter in the SVWI-38 cell line. Competition experiments with an excess of COL1A2 promoter DNA fragment and a constant amount of COL1A2/CAT construct displayed a linear relationship between excess COL1A2 fragment and CAT activity in SVWI-38 cells, suggesting the involvement of a titratable negative effector. Electrophoretic mobility shift assays revealed the presence of a specific DNA-protein complex which was present in SVWI-38 cells and almost absent in control fibroblasts. Methylation interference analysis mapped the region of binding of this factor between nucleotides -80 and -72, relative to the transcription start site. Thus the data presented provide strong evidence for the existence of a negative trans-acting factor that may play a role in the repression of COL1A2 expression in SVWI-38 fibroblasts.

Mice homozygous for the ablm1 mutation show poor viability and depletion of selected B and T cell populations.

The c-abl gene, originally identified as the cellular homolog of the transforming gene of the Abelson murine leukemia virus, encodes a protein-tyrosine kinase of unknown function that is expressed in all mammalian tissues. We have previously described the introduction of a mutation in the c-abl gene into the mouse germline via targeted gene disruption of embryonic stem cells. We now show that mice homozygous for this mutation are severely affected, displaying increased perinatal mortality, runtedness, and abnormal spleen, head, and eye development. We have examined components of the immune system and have found major reductions in B cell progenitors in the adult bone marrow, with less dramatic reductions in developing T cell compartments.

Functional analysis of cis-acting DNA sequences controlling transcription of the human type I collagen genes.

The 3500-base pair region located immediately upstream of the transcriptional start site of the human pro-alpha 2(I) collagen gene contains all the sequences necessary for cell-specific transcription. In transient expression assays, the pro-alpha 2(I) collagen promoter directed the production of high levels of bacterial chloramphenicol acetyltransferase in collagen-producing human fetal fibroblasts. Enzyme activity, on the other hand, was nearly undetectable in extracts from collagen-nonproducing immortalized lymphoblasts. Deletion experiments narrowed the active segment of the human promoter to a phylogenetically conserved sequence comprised between nucleotides-376 and -108, relative to the initiation site of transcription. In similar analyses, the pro-alpha 1(I) collagen gene failed to direct cell-specific transcription. As part of this study, the controversial issue surrounding the putative enhancer element in the first intron of the human pro-alpha 1(I) collagen gene also has been reconsidered. Accordingly, we now propose a more restricted definition of this cis-acting DNA element since its action is exerted in an orientation-preferred manner and with a strong specificity for its own promoter. Moreover, stimulation does not appear to be tissue-specific. Finally, evidence is presented supporting the notion that although structurally different and distinctly arranged, the regulatory sequences of the type I collagen genes may bind similar trans-acting factors.

Molecular pathobiology of human collagens.

The fibril-forming collagens (types I-III, V and XI) represent a homogeneous and evolutionary related group of proteins and genes. In addition to serving as supportive elements, these macromolecules influence the spatial and ontogenic diversity of extracellular matrices, for they regulate a number of developmental programs and cellular activities, such as adhesion, proliferation and migration. Deranged expression of fibrillar collagen genes results in a number of inherited and acquired disorders which greatly affect the structural integrity of the organism. An understanding of collagen biosynthesis and regulation in normal and diseased states provides an opportunity to dissect biological problems which relate to a wide variety of subjects, including morphogenesis, relationships between structure and function of proteins, gene expression and human mutations.

An upstream enhancer and a negative element in the 5' flanking region of the human urokinase plasminogen activator gene.

The 5' flanking region of the human urokinase (uPA) gene has been fused to the reporter chloramphenicol acetyl transferase (CAT) gene and its activity assayed by transfection in two human cell lines. Progressive deletions of the uPA regulatory region from the 5' end maintain a high level of expression provided at least 1870 (in A1251 cells) or 1963 (in HFS10 cells) nucleotides of the 5' flanking region are retained. A DNA fragment from -2350 to -1824 has enhancer properties, stimulating transcription of an enhancerless SV40 early promoter independently of orientation and distance. Internal deletions that still retain the enhancer element reveal the presence of negative cis-acting sequences between -1824 and -1572. Their removal, in fact, increases uPA transcriptional activity. Differences of expression of the uPA-CAT fusion genes in the two cell lines are also observed, indicating the presence of cell-specific cis-acting sequences.

The human urokinase-plasminogen activator gene and its promoter.

The urokinase type of plasminogen activator (uPA) is subject to regulation by hormones, phorbol esters and oncogenic transformation. This enzyme has been suggested to play a key role in processes involving cell migration and tissue remodeling, and to be essential for tumor metastasis. In order to study these processes, we have isolated the human uPA gene, and have determined its entire nucleotide sequence. The gene is organized in 11 exons and is 6.4 kb long. The 5' end of uPA mRNA has been determined by both S1 mapping and primer extension experiments. A fragment of 800 bp containing the entire 5' flanking region shows promoter activity when introduced upstream of a bacterial chloramphenicol acetyltransferase gene and introduced into human cells. The hexanucleotide sequence GGCGGG, previously found at similar regions in several viral and eukaryotic promoters and shown to be essential for promoter activity (McKnight et al. (1984) Cell, 37, 253-262), is repeated three times between the CAAT and the TATA boxes.

High efficiency of replication and expression of foreign genes in SV40-transformed human fibroblasts.

Human fibroblasts (HF) were transformed in vitro with origin-defective SV40 DNA (ori-) using the calcium phosphate co-precipitation technique. The SV40 ori- transformed human cells (HSF) were able to replicate efficiently a recombinant DNA molecule containing the ori sequence of SV40 DNA. Transfection of HFS with pTBC1, a recombinant pi vx plasmid containing the herpes simplex virus thymidine kinase (HSV-TK) gene and the ori SV40 sequences, results in high levels of TK mRNA of correct size. The pTBC1 plasmid does not appear to contain 'poison' sequences and can be efficiently re-established in Escherichia coli after replication in human cells. This host vector system may be of great usefulness in studying the expression of human genes in human cells.