Transgenic expression of human S100A12 induces structural airway abnormalities and limited lung inflammation in a mouse model of allergic inflammation.

BACKGROUND: The calcium-binding protein S100A12 is highly up-regulated in the serum and sputum of patients with allergic asthma and is suggested to be a biomarker and pathologic mediator of asthma. OBJECTIVE: To test the role of S100A12 in mediating airway inflammation in a mouse model of allergic lung inflammation. METHODS: Transgenic (TG) mice that express human S100A12 and wild-type (WT) littermates were sensitized and challenged with ovalbumin (OVA) and assessed for inflammation, lung structure, and function. RESULTS: Following OVA sensitization and challenge, S100A12 TG mice showed reduced peribronchial and perivascular inflammation, mucus production, and eosinophilia as well as attenuated airway responsiveness to contractile agonist compared with WT sensitized and challenged animals. This is explained, at least in part, by remodelled airways in S100A12 TG mice with thinning of the airway smooth muscle. S100A12 exposure induced Fas expression and activation of caspase 3 in cultured airway smooth muscle cells, suggesting that airway smooth muscle abnormalities observed in S100A12 TG mice may be mediated through myocyte apoptosis. CONCLUSION AND CLINICAL RELEVANCE: S100A12 is one of the most abundant proteins found in the airways of human asthmatics, and it was postulated that S100A12 could mediate the inflammatory process. Our study shows for the first time that TG expression of S100A12 in the lung of mice does not exacerbate lung inflammation in a model of OVA-induced allergic inflammation. We speculate that the high levels of S100/calgranulins found in bronchoalveolar lavage fluid of asthmatics and of OVA-treated TG S100A12 mice do not significantly mediate pulmonary inflammation.

Cardiac Protection after Systemic Transplant of Dystrophin Expressing Chimeric (DEC) Cells to the mdx Mouse Model of Duchenne Muscular Dystrophy.

Duchenne Muscular Dystrophy (DMD) is a progressive lethal disease caused by X-linked mutations of the dystrophin gene. Dystrophin deficiency clinically manifests as skeletal and cardiac muscle weakness, leading to muscle wasting and premature death due to cardiac and respiratory failure. Currently, no cure exists. Since heart disease is becoming a leading cause of death in DMD patients, there is an urgent need to develop new more effective therapeutic strategies for protection and improvement of cardiac function. We previously reported functional improvements correlating with dystrophin restoration following transplantation of Dystrophin Expressing Chimeric Cells (DEC) of myoblast origin in the mdx and mdx/scid mouse models. Here, we confirm positive effect of DEC of myoblast (MBwt/MBmdx) and mesenchymal stem cells (MBwt/MSCmdx) origin on protection of cardiac function after systemic DEC transplant. Therapeutic effect of DEC transplant (0.5 × 106) was assessed by echocardiography at 30 and 90 days after systemic-intraosseous injection to the mdx mice. At 90 days post-transplant, dystrophin expression in cardiac muscles of DEC injected mice significantly increased (15.73% ± 5.70 -MBwt/MBmdx and 5.22% ± 1.10 - MBwt/MSCmdx DEC) when compared to vehicle injected controls (2.01% ± 1.36) and, correlated with improved ejection fraction and fractional shortening on echocardiography. DEC lines of MB and MSC origin introduce a new promising approach based on the combined effects of normal myoblasts with dystrophin delivery capacities and MSC with immunomodulatory properties. Our study confirms feasibility and efficacy of DEC therapy on cardiac function and represents a novel therapeutic strategy for cardiac protection and muscle regeneration in DMD.

Creation of Dystrophin Expressing Chimeric Cells of Myoblast Origin as a Novel Stem Cell Based Therapy for Duchenne Muscular Dystrophy.

Over the past decade different stem cell (SC) based approaches were tested to treat Duchenne Muscular Dystrophy (DMD), a lethal X-linked disorder caused by mutations in dystrophin gene. Despite research efforts, there is no curative therapy for DMD. Allogeneic SC therapies aim to restore dystrophin in the affected muscles; however, they are challenged by rejection and limited engraftment. Thus, there is a need to develop new more efficacious SC therapies. Chimeric Cells (CC), created via ex vivo fusion of donor and recipient cells, represent a promising therapeutic option for tissue regeneration and Vascularized Composite Allotransplantation (VCA) due to tolerogenic properties that eliminate the need for lifelong immunosuppression. This proof of concept study tested feasibility of myoblast fusion for Dystrophin Expressing. Chimeric Cell (DEC) therapy through in vitro characterization and in vivo assessment of engraftment, survival, and efficacy in the mdx mouse model of DMD. Murine DEC were created via ex vivo fusion of normal (snj) and dystrophin-deficient (mdx) myoblasts using polyethylene glycol. Efficacy of myoblast fusion was confirmed by flow cytometry and dystrophin immunostaining, while proliferative and myogenic differentiation capacity of DEC were assessed in vitro. Therapeutic effect after DEC transplant (0.5 × 106) into the gastrocnemius muscle (GM) of mdx mice was assessed by muscle functional tests. At 30 days post-transplant dystrophin expression in GM of injected mdx mice increased to 37.27 ± 12.1% and correlated with improvement of muscle strength and function. Our study confirmed feasibility and efficacy of DEC therapy and represents a novel SC based approach for treatment of muscular dystrophies.

The myeloid-cell-specific c-fes promoter is regulated by Sp1, PU.1, and a novel transcription factor.

The protein product of the c-fps/fes (c-fes) proto-oncogene has been implicated in the normal development of myeloid cells (macrophages and neutrophils). mRNA for c-fes has been detected exclusively in myeloid cells and vascular endothelial cells in adult mammals. Although a 13-kilobase-pair (kb) human c-fes transgene exhibits high levels of expression in mice, the sequences that confer myeloid-cell-specific expression of the human c-fes gene have not been defined. Transient-transfection experiments demonstrated that plasmids containing 446 bp of c-fes 5'-flanking sequences linked to a luciferase reporter gene were active exclusively in myeloid cells. No other DNA element within the 13-kb human c-fes locus contained positive cis-acting elements, with the exception of a weakly active region within the 3'-flanking sequences. DNase I footprinting assays revealed four distinct sites that bind myeloid nuclear proteins (-408 to -386, -293 to -254, -76 to -65, and -34 to +3). However, the first two footprints resided in sequences that were largely dispensable for transient activity. Plasmids containing 151 bp of 5'-flanking sequences confer myeloid-cell-specific gene expression. Electrophoretic mobility shift analyses demonstrated that the 151-bp region contains nuclear protein binding sites for Sp1, PU.1, and/or Elf-1, and a novel factor. This unidentified factor binds immediately 3' of the PU.1/Elf-1 sites and appears to be myeloid cell specific. Mutation of the PU.1/Elf-1 site or the 3' site (FP4-3') within the context of the c-fes promoter resulted in substantially reduced activity in transient transfections. Furthermore, transient-cotransfection assay demonstrated that PU.1 (and not Elf-1) can transactivate the c-fes promoter in nonmyeloid cell lines. We conclude that the human c-fes gene contains a strong myeloid-cell-specific promoter that is regulated by Sp1, PU.1, and a novel transcription factor.

The E1A oncogene induces resistance to the effects of 1,25-dihydroxyvitamin D3 on inhibition of growth of mouse keratinocytes.

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] inhibited DNA synthesis in transformed mouse keratinocytes (Pam212) in a time- and dose-dependent manner as measured by [3H]thymidine incorporation. To investigate the mechanism through which 1,25-(OH)2D3 acts, we examined its effects on Pam212 cells further transformed with the E1A oncogene. Here, we show that transformation of the cells with the E1A oncogene induced resistance to the effects of 1,25-(OH)2D3 on inhibition of growth of Pam212 cells. While 1,25-(OH)2D3 treatment increased the level of expression of vitamin D receptor mRNA 20-fold in parental cells, the E1A-transformed cells failed to express vitamin D receptor mRNA even after treatment with 1,25-(OH)2D3. Transfection of the E1A-transformed cell line with an expression construct encoding the vitamin D receptor restored receptor expression as well as the inhibition of growth by 1,25-(OH)2D3. These results suggest that one of the mechanisms for acquisition of 1,25-(OH)2D3 resistance induced by E1A may involve loss of vitamin D receptor inducibility by 1,25-(OH)2D3.

Transforming growth factor beta 1 stimulates type II collagen expression in cultured periosteum-derived cells.

Chondrogenesis can occur during a bone repair process, which is related to several growth factors. Transforming growth factor beta 1 (TGF-beta 1) downregulates the expression of type II collagen by chondrocytes in vitro, but injection of TGF-beta 1 into the periosteum in vivo increases type II collagen mRNA levels and initiates chondrogenesis. We examined the effect of TGF-beta 1 on collagen gene expression in a bovine periosteum-derived cell culture system to evaluate its direct effect on the periosteum. Cultured cells expressed alkaline phosphatase and collagen pro alpha 1(I) and pro alpha 1(II) mRNAs. A low level of type II collagen synthesis was demonstrated by immunoprecipitation. TGF-beta 1 had no effect on periosteal cell proliferation. Expression of collagen pro alpha 1(I) mRNA did not change with TGF-beta 1 treatment, but alkaline phosphatase mRNA showed a dose-dependent decrease. Expression of collagen pro alpha 1(II) mRNA was stimulated 2.7-fold by TGF-beta 1. TGF-beta 1 also caused a 2.6-fold increase in type II collagen synthesis by immunoprecipitation. These findings indicate that TGF-beta 1 is an enhancer of the expression of the chondrocyte phenotype of the periosteal cells and suggest that TGF-beta 1 is important in initiating and promoting cartilage formation in vivo.

Regulatory regions from the Brn4 promoter direct LACZ expression to the developing forebrain and neural tube.

To characterize cis-acting regulatory elements of the mouse POU-domain gene, Brain-4/Pou3F4, transgenic mouse pedigrees were generated that contained the LacZ reporter gene under the control of Brn4 5' flanking sequences. A six kilobase promoter region was identified that reproducibly directed expression of the reporter gene to the forebrain and neural tube of developing mouse embryos. Deletional analysis of this promoter region indicates that at least two positive cis-active elements can direct expression to the developing neural tube. These data characterize a transgenic promoter region that will be useful in directing expression to the developing neural tube during the ontogeny of the forebrain.

Regulation of the expression of the type-II collagen gene in periosteum-derived cells by three members of the transforming growth factor-beta superfamily.

Although transforming growth factors-beta and bone morphogenetic proteins are both capable of inducing bone formation in vivo, the target cells of their osteoinductive actions may be different. To evaluate periosteal cells as potential targets of the actions of transforming growth factor-beta and bone morphogenetic protein, we investigated the ability of three members of the transforming growth factor-beta superfamily to modulate expression of the gene encoding the alpha 1(II) chain of type-II collagen in periosteum-derived cells in vitro. The results demonstrate that transforming growth factor-beta mRNA is expressed by periosteum-derived cells and that exogenous transforming growth factor-beta 1 acts to upregulate expression of the gene encoding collagen alpha 1(II). This effect was observed as early as 12 hours after administration of transforming growth factor-beta 1 but was not observed in response to bone morphogenetic proteins 3 or 4. No synergy was demonstrated between transforming growth factor-beta 1 and bone morphogenetic protein-3 in the ability to upregulate expression of the collagen alpha 1(II) gene. These results support the hypothesis that committed periosteal mesenchymal cells are cellular targets of the action of transforming growth factor-beta.

Acidic fibroblast growth factor (aFGF) injection stimulates cartilage enlargement and inhibits cartilage gene expression in rat fracture healing.

The effect of the administration of acidic fibroblast growth factor (aFGF) on normal fracture healing was examined in a rat fracture model. One microgram of aFGF was injected into the fracture site between the first and the ninth day after fracture either every other day or every day. aFGF-injected calluses were significantly larger than control calluses, although this does not imply an increased mechanical strength of the callus. Histology showed a marked increase in the size of the cartilaginous soft callus. Total DNA and collagen content in the cartilaginous portion of the aFGF-injected calluses were greater than those of controls, although the collagen content/DNA content ratio was not different between the aFGF-injected and control calluses. Fracture calluses injected with aFGF remained larger than controls until 4 weeks after fracture. The enlarged cartilaginous portion of the aFGF-injected calluses seen at 10 days after fracture was replaced by trabecular bone at 3 and 4 weeks. Northern blot analysis of total cellular RNA extracted separately from the cartilaginous soft callus and the bony hard callus showed decreased expression of type II procollagen and proteoglycan core protein mRNA in the aFGF-injected calluses when compared with controls. A slight decrease in types I and III procollagen mRNA expression was also observed. We concluded that aFGF injections induced cartilage enlargement and decreased mRNA expression for type II procollagen and proteoglycan core protein.

Changes in the subcellular localization of the Brn4 gene product precede mesenchymal remodeling of the otic capsule.

To better understand the genetic mechanisms that regulate the formation of the temporal bone, we have characterized the developmental expression pattern of the mouse gene, Brn4/Pou3f4, which plays a central role in bony labyrinth formation. Expression of this gene is initially detected in the ventral aspect of the otic capsule at 10.5 days post coitus (dpc), and correlates with the onset of mesenchymal condensation in the otic capsule. As the otic capsule condenses further and surrounds the entire otic vesicle, the Brn4 gene product is detected throughout the inner ear in the mesenchyme of both the cochlear and vestibular aspects. Early in otic embryogenesis, the Brn4 gene product is localized to the nucleus of the vast majority of cells in which it is expressed. The Brn4 gene product remains nuclear in those regions of the otic capsule that eventually give rise to the mature bony labyrinth. However, the subcellular localization of the Brn4 gene product shifts from strictly nuclear to perinuclear in those regions of the otic capsule that will cavitate to form acellular regions in the temporal bone, such as the scala tympani, scala vestibuli, and the internal auditory meatus. These data provide a detailed analysis of the expression pattern of the Brn4 gene, and provide insight into the role of the Brn4 gene product and its regulation during otic capsule formation.

Isolation and analysis of ribonucleic acids from skeletal tissues.

We report a method for the isolation of total cellular RNA from mineralized or cartilaginous tissues. The procedure accommodates the large amount of hydroxyapatite and high buoyant density proteoglycans present in skeletal tissue samples, as well as the low cell density characteristic of these tissues. The procedure can be reliably used for processing a large number of small (100-800 mg) tissue samples. Tissues are homogenized in guanidine hydrochloride solution, then centrifuged at low speed, and filtered to remove the nonsolubilized extracellular matrix proteins. Subsequent high speed density gradient centrifugation produces a high yield of RNA (0.2-0.6 micrograms RNA/mg tissue) which is precipitated in a low pH sodium acetate solution. RNA extracted by this method has been analyzed for the expression of various genes by Northern blotting. In addition to mRNAs of bone- and cartilage-specific proteins, messenger RNA for growth factors, proto-oncogenes, and heat shock proteins can be detected.

Expression of two myeloid cell-specific genes requires the novel transcription factor, c-fes expression factor.

The protein product of the c-fes proto-oncogene has been implicated in the normal development of myeloid cells (macrophages and granulocytes). We have previously shown that 151 base pairs of c-fes 5'-flanking sequences are sufficient for myeloid cell-specific expression and include functional binding sites for Sp1, PU.1, and a novel nuclear factor (Heydemann, A., Juang, G., Hennessy, K., Parmacek, M. S., and Simon, M. C. (1996) Mol. Cell. Biol. 16, 1676-1686). This novel hematopoietic transcription factor, termed FEF (c-fes expression factor), binds to a cis-acting element that is located at nucleotides -9 to -4 of the c-fes promoter between two Ets binding sites (at -19 to -15 and -4 to +1) which bind PU.1. We now show that a FEF binding site exists in the myeloid cell-specific regulatory region of a second gene, the -2.7-kilobase pair enhancer of chicken lysozyme. The lysozyme FEF site is immediately 5' to a PU. 1 site, analogous to their arrangement in the c-fes promoter, and allows the formation of a preliminary FEF consensus site, 5'-GAAT(C/G)A-3'. This consensus site does not match any sites for known transcription factors. Importantly, although PU.1 binds immediately 3' of the FEF site in both the c-fes promoter and the chicken lysozyme enhancer (CLE), we show that they bind independently. The FEF sites are required for high levels of transcription by both the CLE and the c-fes promoter in transient transfection experiments. Importantly, elimination of the CLE FEF site abolishes all transcriptional activity of this enhancer element. Mutation of the adjacent PU.1 site in either the c-fes promoter or the CLE, reduces activity by approximately 50%. Therefore, transcription of both lysozyme and fes in myeloid cells requires FEF and PU.1. UV cross-linking experiments show that the FEF binding activity consists of a single 70-kDa protein in both human and murine cell lines. FEF binding activity is not affected by antibodies that specifically recognize a number of cloned transcription factors. Collectively, these data indicate that we have identified a novel transcription factor that is functionally important for the expression of at least two myeloid cell-specific genes.

Cardiomyopathy in animal models of muscular dystrophy.

Arrhythmia and cardiomyopathy frequently accompany muscular dystrophy. In the last year, the cardiovascular consequences of muscular dystrophy gene mutations have been established through studies of murine models. These models have highlighted the potential role of primary defects in cardiac muscle as well as those secondary cardiovascular outcomes that arise from severe muscle disease. This review focuses on three areas. Recent studies using mouse models have shown that the dystrophin-associated proteins, the sarcoglycans and alpha-dystrobrevin, are critical for both cardiac and skeletal muscle membrane function, yet may exert their roles by different molecular mechanisms. New findings have shown that cytoskeletal proteins at the nuclear membrane, such as emerin and lamin AC, cause muscular dystrophy and cardiomyopathy with cardiac conduction system disease. Finally, the mechanism of cardiac and muscle degeneration in myotonic dystrophy has been re-evaluated through a series of studies using murine models. Implications for human therapy are considered in light of these new findings.

Estrogen receptor mRNA expression in callus during fracture healing in the rat.

Estrogen has profound effects on bone metabolism, yet its precise mechanism of action remains unknown. Several recent investigations have located the estrogen receptor (ER) on osteoblast-like cells, suggesting a potential direct action of estrogen via its own receptor on bone cells. The protective role of estrogen on bone in osteoporosis is well known; however, neither the existence nor the mechanism of an estrogenic role in fracture healing has been well studied. In this investigation we used a modification of polymerase chain reaction (PCR) amplification to detect and quantify ER messenger RNA (mRNA) levels in rat fracture callus previously undetectable by northern analysis. Verification of correctly amplified cDNA fragments was provided by restriction digestion. PCR failed to detect ER mRNA in total RNA extracts from tendon and fibrocartilage. The ER message was present in fracture callus at levels up to 70% of that found in rat uterus, and demonstrated a transient 13-fold increase in transcription by day 14 with return to baseline by day 31 of fracture healing. These data demonstrate that the rat estrogen receptor gene is expressed in fracture callus and suggest that estrogen may play an important role in the normal fracture healing process. In addition, this work adds to the growing body of evidence which supports a possible direct action of estrogen via its receptor on osteogenic cells.

A minimal c-fes cassette directs myeloid-specific expression in transgenic mice.

The c-fes proto-oncogene encodes a 92-kd protein tyrosine kinase whose expression is restricted largely to myeloid and endothelial cells in adult mammals. A 13.2-kilobase (kb) human c-fes genomic fragment was previously shown to contain cis-acting element(s) sufficient for a locus control function in bone marrow macrophages. Locus control regions (LCRs) confer transgene expression in mice that is integration site independent, copy number dependent, and similar to endogenous murine messenger RNA levels. To identify sequences required for this LCR, c-fes transgenes were analyzed in mice. Myeloid-cell-specific, deoxyribonuclease-I-hypersensitive sites localized to the 3' boundary of exon 1 and intron 3 are required to confer high-level transgene expression comparable to endogenous c-fes, independent of integration site. We define a minimal LCR element as DNA sequences (nucleotides +28 to +2523 relative to the transcription start site) located within intron 1 to intron 3 of the human locus. When this 2.5-kb DNA fragment was linked to a c-fes complementary DNA regulated by its own 446-base-pair promoter, integration-site-independent, copy-number-dependent transcription was observed in myeloid cells in transgenic mice. Furthermore, this 2.5-kb cassette directed expression of a heterologous gene (enhanced green fluorescent protein) exclusively in myeloid cells. The c-fes regulatory unit represents a novel reagent for targeting gene expression to macrophages and neutrophils in transgenic mice.

TGF-beta 1 prevents hypertrophy of epiphyseal chondrocytes: regulation of gene expression for cartilage matrix proteins and metalloproteases.

Using an in vitro model of rat epiphyseal chondrocyte differentiation in which cells are maintained in a three-dimensional cell pellet, we show that exogenous TGF-beta 1 reversibly prevents terminal differentiation of epiphyseal chondrocytes into hypertrophic cells. Through maintenance of gene expression for the cartilage matrix proteins type II collagen and aggrecan core protein, and with coordinate inhibition of expression of genes encoding the metalloproteases collagenase and stromelysin, TGF-beta 1 stabilizes the phenotype of the prehypertrophic epiphyseal chondrocyte. This ability of TGF-beta 1 to stabilize the cartilage phenotype is critically dependent on culture conditions. Epiphyseal chondrocytes cultured as a subconfluent monolayer of cells dedifferentiate (reduce type II collagen and aggrecan core protein expression, increase metalloprotease expression, and acquire a spindled morphology) in response to short-term TGF-beta 1 treatment. Increasing the initial seeding density and allowing the cells to become multilayered prior to the addition of growth factor reverse the effects of TGF-beta 1 on type II collagen and transin/stromelysin gene expression and maintain a rounded cellular morphology. This finding emphasizes the importance of considering cell density and environmental context in the analysis of the regulatory action of peptide growth factors in general and of the TGF-beta s in particular. We propose that one function of TGF-beta 1 during endochondral ossification is regulation of chondrocyte growth and differentiation through modulation of the relative expression of cartilage matrix proteins and metalloproteases. This function of TGF-beta 1 helps illustrate how the regulation of diverse cellular processes such as matrix synthesis, matrix degradation, and cell growth and differentiation may be coordinated at the molecular level by a single peptide growth factor.

Inhibition of the chondrocyte phenotype by retinoic acid involves upregulation of metalloprotease genes independent of TGF-beta.

Retinoic acid has been identified as a key morphogen governing pattern formation in the developing cartilaginous skeleton. Retinoids have also been implicated in the premature closure of the cartilage growth plate following vitamin A intoxication or administration of retinoids for dermatologic conditions. Previous studies of the mechanism of action of retinoids in non-chondrogenic cells have concluded that retinoic acid is a negative regulator of AP-1 responsive metalloprotease genes. We show that inhibition of expression of the cartilage phenotype by retinoic acid in epiphyseal chondrocytes is associated with positive regulation of AP-1 responsive metalloprotease genes, as well as induction of gene expression for the two components of the transcription factor AP-1, c-fos and c-jun. Despite the similar effects of TGF-beta 1 on expression of cartilage matrix proteins and metalloproteases in this culture system, no appreciable changes in the expression of TGF-beta isoforms were evident in response to retinoic acid treatment. The present investigation demonstrates that regulation of AP-1 responsive genes by retinoic acid can be either positive or negative, depending on the target cell type, and illuminates new mechanisms by which retinoic acid and other retinoids may exert control during development and growth of the limb.