Mouse retinal development: Role of cell adhesion and mechanism of gene regulation

Cell differentiation and pattern formation are fundamental processes in animal development that are under intense investigation. The mouse retina is a good model to study these processes because it has seven distinct cell types, and three well-laminated nuclear layers that form during embryonic and postnatal life. β-catenin functions as both the nuclear effector for the canonical Wnt pathway and a cell adhesion molecule, and is required for the development of various organs. To study the function of β-catenin in retinal development, I used a Cre-loxP system to conditionally ablate β-catenin in the developing retina. Deletion of β-catenin led to disrupted laminar structure but did not affect the differentiation of any of the seven cell types. Eliminating β-catenin did not reduce progenitor cell proliferation, although enhanced apoptosis was observed. Further analysis showed that disruption of cell adhesion was the major cause of the observed patterning defects. Overexpression of β-catenin during retinal development also disrupted the normal retinal lamination and caused a transdifferentiation of neurons into pigmented cells. The results indicate that β-catenin functions as a cell adhesion molecule but not as a Wnt pathway component during retinal neurogenesis, and is essential for lamination but not cell differentiation. The results further imply that retinal lamination and cell differentiation are genetically separable processes. ^ Sonic hedgehog (shh) is expressed in retinal ganglion cells under the control of transcription factor Pou4f2 during retinal development. Previous studies identified a phylogenetically conserved region in the first intron of shh containing a Pou4f2 binding site. Transgenic reporter mice in which reporter gene expression was driven by this region showed that this element can direct gene expression specifically in the retina, but expression was not limited to the ganglion cells. From these data I hypothesized that this element is required for shh expression in the retina but is not sufficient for specific ganglion cell expression. To further test this hypothesis, I created a conditional allele by flanking this region with two loxP sites. Lines carrying this allele will be crossed with retinal-specific Cre lines to remove this element in the retina. My hypothesis predicts that alteration in shh expression and subsequent retinal defects will occur in the retinas of these mice. ^

A Novel Mechanism of Skin Tumor Promotion Involving Interferon-gamma (IFNγ)/Signal Transducer and Activator of Transcription-1 (Stat1) Signaling in Epidermis

The JAK-STAT pathway is a major signaling pathway involved in many biological processes including proliferation, apoptosis, and differentiation. Aberrant expression of STATs has been reported in multiple human cancers and murine mouse models of tumorigenesis. Previous studies from our lab and others have established a critical role for Stat3 in epithelial tumorigenesis, but the role of Stat1 is largely unknown. The current study was designed to explore the role of Stat1 during multistage skin carcinogenesis. Topical treatment with both TPA and the anthrone derivative chrysarobin (CHRY) led to rapid phosphorylation of Stat1 on both tyrosine (Tyr701) and serine (Ser727) residues in epidermis. CHRY treatment also led to upregulation of unphosphorylated Stat1 (uStat1) at later time points. In addition, CHRY treatment also led to upregulation of IRF-1 mRNA and protein which was dependent on Stat1. Further analyses demonstrated that topical treatment with CHRY but not TPA upregulated interferon-gamma (IFNg) mRNA in the epidermis and that the induction of both IRF-1 and uStat1 was dependent on IFNg signaling. Stat1 deficient (Stat1-/-) mice were highly resistant to skin tumor promotion by CHRY. In contrast, the tumor response (in terms of both papillomas and squamous cell carcinomas) was similar in Stat1-/- mice and wild-type littermates with TPA as the promoter. Histological evaluation of the proliferative response confirmed the data obtained from the tumor study for both TPA and CHRY. In addition, maximal induction of both cyclooxygenase-2 and inducible nitric oxide synthase in epidermis following treatment with CHRY was also dependent on the presence of functional Stat1. Following CHRY treatment, Stat1-/- mice exhibited reduced macrophage infiltration and reduced production of many immune cell derived chemokines/cytokines. These studies define a novel mechanism associated with skin tumor promotion by the anthrone class of tumor promoters involving upregulation of IFNg signaling in the epidermis and downstream signaling through activated (phosphorylated) Stat1 and subsequent upregulation of IRF-1 and uStat1.

Delayed Thrombus Resolution and Fibroproliferative Vascular Wound Healing From Deficiency of Type III Collagen: a Paradoxical Mechanism for Tissue Fragility

Vascular Ehlers-Danlos syndrome is a heritable disease of connective tissue caused by mutations in COL3A1, conferring a tissue deficiency of type III collagen. Cutaneous wounds heal poorly in these patients, and they are susceptible to spontaneous and catastrophic rupture of expansible hollow organs like the gut, uterus, and medium-sized to large arteries, which leads to premature death. Although the predisposition for organ rupture is often attributed to inherent tissue fragility, investigation of arteries from a haploinsufficient Col3a1 mouse model (Col3a1+/-) demonstrates that mutant arteries withstand even supraphysiologic pressures comparably to wild-type vessels. We hypothesize that injury that elicits occlusive thrombi instead unmasks defective thrombus resolution resulting from impaired production of type III collagen, which causes deranged remodeling of matrix, persistent inflammation, and dysregulated behavior by resident myofibroblasts, culminating in the development of penetrating neovascular channels that disrupt the mechanical integrity of the arterial wall. Vascular injury and thrombus formation following ligation of the carotid artery reveals an abnormal persistence and elevated burden of occlusive thrombi at 21 post-operative days in vessels from Col3a1+/- mice, as opposed to near complete resolution and formation of a patent and mature neointima in wild-type mice. At only 14 days, both groups harbor comparable burdens of resolving thrombi, but wild-type mice increase production of type III collagen in actively resolving tissues, while mutant mice do not. Rather, thrombi in mutant mice contain higher burdens of macrophages and proliferative myofibroblasts, which persist through 21 days while wild-type thrombi, inflammatory cells, and proliferation all regress. At the same time that increased macrophage burdens were observed at 14 and 21 days post ligation, the medial layer of mutant arterial walls concurrently harbored a significantly higher incidence of penetrating neovessels compared with those in wild-type mice. To assess whether limited type III collagen production alters myofibroblast behavior, fibroblasts from vEDS patients with COL3A1 missense mutations were seeded into three-dimensional fibrin gel constructs and stimulated with transforming growth factor-β1 to initiate myofibroblast differentiation. Although early signaling events occur similarly in all cell lines, late extracellular matrix- and mechanically-regulated events like transcriptional upregulation of type I and type III collagen secretion are delayed in mutant cultures, while transcription of genes encoding intracellular contractile machinery is increased. Sophisticated imaging of collagen synthesized de novo by resident myofibroblasts visualizes complex matrix reorganization by control cells but only meager remodeling by COL3A1 mutant cells, concordant with their compensatory contraction to maintain tension in the matrix. Finally, administration of immunosuppressive rapamycin to mice following carotid ligation sufficiently halts the initial inflammatory phase of thrombus resolution and fully prevents both myofibroblast migration into the thrombus and the differential development of neovessels between mutant and wild-type mice, suggesting that pathological defects in mutant arteries develop secondarily to myofibroblast dysfunction and chronic inflammatory stimulation, rather than as a manifestation of tissue fragility. Together these data establish evidence that pathological defects in the vessel wall architecture develop in mutant arteries as sequelae to abnormal healing and remodeling responses activated by arterial injury. Thus, these data support the hypothesis that events threatening the integrity of type III collagen-deficient vessels develop not as a result of inherent tissue weakness and fragility at baseline but instead as an episodic byproduct of abnormally persistent granulation tissue and fibroproliferative intravascular remodeling.

Investigation of the mechanism of increased melanoma incidence in UV -irradiated mouse skin

The availability of transplantable, syngeneic murine melanomas made it possible to study the potential effects of UV radiation on the growth and progression of melanomas in an animal model. The purpose of my study was to determine how UV-irradiation increases the incidence of melanoma out-growth, when syngeneic melanoma cells are transplanted into a UV-irradiated site. Short term intermittent UVB exposure produces a transitory change in the mice which allows the increased outgrowth of melanoma cells injected into the UV-irradiated site. One possible mechanism is an immunomodulatory effect of UVR on the host. An alternative mechanism to account for the increased tumor incidence in the UV-irradiated site, is the release of inflammatory mediators from UV-irradiated epidermal cells. A third possibility is that UVR could induce the production and/or release of melanoma-specific growth factors resulting in increased melanoma outgrowth.^ My first step in distinguishing among these different possible mechanisms was to characterize further the conditions leading to increased development of melanoma cells in UV-irradiated mouse skin. Next, I attempted to determine which of the 3 proposed mechanisms was most likely. To do this, I defined the specificity of the effect by examining the growth of additional C3H tumorigenic cell lines in UV-irradiated skin. Second, I determined the immunogenicity of these tumor cell lines. The tumor cell lines exhibiting increased tumor incidence are restricted to those tumor cell lines which are immunogenic in normal C3H mice. Third, I determined the effect of UVR on melanoma development did not occur in immunosuppressed mice.^ Because of results from these three lines of investigation suggested that the effect was immunologically mediated, I then investigated whether specific immune reactions were affected by local UV irradiation. To accomplish this, I investigated the effect of UVR on cutaneous immune cells and on induction of contact hypersensitivity (CHS), and I also determined the effect of UVR on the development and the expression of systemic immunity against the melanoma cells. There is no clear cut relationship between the number of Langerhans or Thy1+ cells and the UV effect on tumor incidence. Furthermore, there was no suppression of CHS in the UV-irradiated mice. While the development of systemic immunity is significantly reduced, it appears to be sufficient to provide in vivo immunity to tumor challenge. However the elicitation of tumor immunity in immunized mice can be abrogated if tumor challenge occurs in the site of UV irradiation. This investigation provides new information on an effect of UVR on the elicitation of tumor immunity. Furthermore, it indicates that UV radiation can play a role in the development of melanoma other than just in the transformation of melanocytes. ^

Mechanism of subunit interaction and DNA binding of the heterotrimeric CCAAT binding factor CBF

Many eukaryotic promoters contain a CCAAT element at a site close ($-$80 to $-$120) to the transcription initiation site. CBF (CCAAT Binding Factor), also called NF-Y and CP1, was initially identified as a transcription factor binding to such sites in the promoters of the Type I collagen, albumin and MHC class II genes. CBF is a heteromeric transcription factor and purification and cloning of two of the subunits, CBF-A and CBF-B revealed that it was evolutionarily conserved with striking sequence identities with the yeast polypeptides HAP3 and HAP2, which are components of a CCAAT binding factor in yeast. Recombinant CBF-A and CBF-B however failed to bind to DNA containing CCAAT sequences. Biochemical experiments led to the identification of a third subunit, CBF-C which co-purified with CBF-A and complemented the DNA binding of recombinant CBF-A and CBF-B. We have recently isolated CBF-C cDNAs and have shown that bacterially expressed purified CBF-C binds to CCAAT containing DNA in the presence of recombinant CBF-A and CBF-B. Our experiments also show that a single molecule each of all the three subunits are present in the protein-DNA complex. Interestingly, CBF-C is also evolutionarily conserved and the conserved domain between CBF-C and its yeast homolog HAP5 is sufficient for CBF-C activity. Using GST-pulldown experiments we have demonstrated the existence of protein-protein interaction between CBF-A and CBF-C in the absence of CBF-B and DNA. CBF-B on other hand, requires both CBF-A and CBF-C to form a ternary complex which then binds to DNA. Mutational studies of CBF-A have revealed different domains of the protein which are involved in CBF-C interaction and CBF-B interaction. In addition, CBF-A harbors a domain which is involved in DNA recognition along with CBF-B. Dominant negative analogs of CBF-A have also substantiated our initial observation of assembly of CBF subunits. Our studies define a novel DNA binding structure of heterotrimeric CBF, where the three subunits of CBF follow a particular pathway of assembly of subunits that leads to CBF binding to DNA and activating transcription. ^

Mechanism of silica -induced apoptosis in human alveolar macrophages

The mechanisms involved in the development of pulmonary silicosis have not been well defined, however most current evidence implicates a central role for alveolar macrophages in this process. We propose that the fibrotic potential of a particulate depends upon its ability to cause apoptosis in alveolar macrophage (AM). The overall goal of this study was to determine the mechanism of silica-induced apoptosis of AM. Human AM were treated with fibrogenic, poorly fibrogenic and nonfibrogenic model particulates, such as, silica, amorphous silica and titanium dioxide, respectively (equal surface area). Treatment with silica resulted in apoptosis in human AM as observed by morphology, DNA fragmentation and Cell Death ELISA assays. In contrast, amorphous silica and titanium dioxide demonstrated no significant apoptotic potential. To elucidate the possible mechanism by which silica causes apoptosis, we investigated the role of the scavenger receptor (SR) in silica-induced apoptosis. Cells were pretreated with and without SR ligand binding inhibitors, polyinosinic acid (Poly I), fucoidan and high density lipoprotein (HDL), prior to silica treatment. Pretreatment with Poly I and fucoidan resulted in significant inhibition of silica-induced apoptosis suggesting that silica-induced AM apoptosis is mediated via the SR. Further, we examined the involvement of interleukin converting enzyme (ICE) family of proteases in silica-mediated apoptosis. Silica activated ICE, Ich-1L, cpp32 beta and cleavage of PARP. Taken together, these results suggested that (1) fibrogenic particulates, such as, silica caused apoptosis of alveolar macrophages, (2) this apoptotic potential of fibrogenic particulates may be a critical factor in initiating an inflammatory response resulting in fibrosis, (3) silica-induced apoptosis of alveolar macrophages may be due to the interaction of silica particulates with the SR, and (4) silica-induced apoptosis involves the activation of the ICE family of proteases. An understanding of the molecular events involved in fibrogenic particulate-induced apoptosis may provide a useful insight into the mechanism involved in particulate-induced fibrosis. ^

Analysis of the mechanism of replication inhibition of the tumor suppressor gene p53

p53 plays a role in cell cycle arrest and apoptosis. p53 has also been shown to be involved in DNA replication. To study the effect of p53 on DNA replication, we utilized a SV40 based shuttle vector system. The pZ402 shuttle vector, was constructed with a mutated T-antigen unable to interact with p53 but able to support replication of the shuttle vector. When a transcriptional activation domain p53 mutant was tested for its ability to inhibit DNA replication no inhibition was observed. Competition assays with the DNA binding domain of p53 was also able to block the inhibition of DNA replication by p53 suggesting that p53 can inhibit DNA replication through the transcriptional activation of a target gene. One likely target gene, p21$\sp{\rm cip/waf}$ was tested to determine whether p53 inhibited DNA replication by transcriptionally activating p21$\sp{\rm cip/waf}$. Two independent approaches utilizing p21$\sp{\rm cip/waf}$ null cells or the expression of an anti-sense p21$\sp{\rm cip/waf}$ expression vector were utilized. p53 was able to inhibit pZ402 replication independently of p21$\sp{\rm cip/waf}$. p53 was also able to inhibit DNA replication independent of the p53 target genes Gadd45 and the replication processivity factor PCNA. The inhibition of DNA replication by p53 was also independent of direct DNA binding to a consensus site on the replicating plasmid. p53 mutants can be classified into two categories: conformational and DNA contact mutants. The two types of p53 mutants were tested for their effects on DNA replication. While all conformational mutants were unable to inhibit DNA replication three out of three DNA contact mutants tested were able to inhibit DNA replication. The work here studies the effect wild-type and mutant p53 has on DNA replication and demonstrated a possible mechanism by which wild-type p53 could inhibit DNA replication through the transcriptional activation of a target gene. ^

Mechanism of molecular regulation of nuclear -encoded mitochondrial gene expression by electrical stimulation in the cultured neonatal rat cardiac myocytes

To answer the question whether increased energy demand resulting from myocyte hypertrophy and enhanced $\beta$-myosin heavy chain mRNA, contractile protein synthesis and assembly leads to mitochondrial proliferation and differentiation, we set up an electrical stimulation model of cultured neonatal rat cardiac myocytes. We describe, as a result of increased contractile activity, increased mitochondrial profiles, cytochrome oxidase mRNA, and activity, as well as a switch in mitochondrial carnitine palmitoyltransferase-I (CPT-I) from the liver to muscle isoform. We investigate physiological pathways that lead to accumulation of gene transcripts for nuclear encoded mitochondrial proteins in the heart. Cardiomyocytes were stimulated for varying times up to 72 hr in serum-free culture. The mRNA contents for genes associated with transcriptional activation (c-fos, c-jun, junB, nuclear respiratory factor 1 (Nrf-1)), mitochondrial proliferation (cytochrome c (Cyt c), cytochrome oxidase), and mitochondrial differentiation (carnitine palmitonyltransferase I (CPT-I) isoforms) were measured. The results establish a temporal pattern of mRNA induction beginning with c-fos (0.25-3 hr) and followed by c-jun (0.5-3 hr), junB (0.5-6 hr), NRF-1 (1-12 hr), Cyt c (12-72 hr), cytochrome c oxidase (12-72 hr). Induction of the latter was accompanied by a marked decrease in the liver-specific CPT-I mRNA. Electrical stimulation increased c-fos, $\beta$-myosin heavy chain, and Cyt c promoter activities. These increases coincided with a rise in their respective endogenous gene transcripts. NRF-1, cAMP response element (CRE), and Sp-1 site mutations within the Cyt c promoter reduced luciferase expression in both stimulated and nonstimulated myocytes. Mutations in the Nrf-1 and CRE sites inhibited the induction by electrical stimulation or by transfection of c-jun into non-paced cardiac myocytes whereas mutation of the Sp-1 site maintained or increased the fold induction. This is consistent with the appearance of NRF-1 and fos/jun mRNAs prior to that of Cyt c. Overexpression of c-jun by transfection also activates the Nrf-1 and Cyt c mRNA sequentially. Electrical stimulation of cardiac myocytes activates the c-Jun-N-terminal kinase so that the fold-activation of the cyt c promoter is increased by pacing when either c-jun or c-fos/c-jun are cotransfected. We have identified physical association of Nrf-1 protein with the Nrf-1 enhancer element and of c-Jun with the CRE binding sites on the Cyt c promoter. This is the first demonstration that induction of Nrf-1 and c-Jun by pacing of cardiac myocytes directly mediates Cyt c gene expression and mitochondrial proliferation in response to hypertrophic stimuli in the heart.^ Subsequent to gene activation pathways that lead to mitochondrial proliferation, we observed an isoform switch in CPT-I from the liver to muscle mRNA. We have found that the half-life for the muscle CPT-I is not affected by electrical stimulation, but electrical decrease the T1/2 in the liver CPT-I by greater than 50%. This suggests that the liver CPT-I switch to muscle isoform is due to (1) a decrease in T1/2 of liver CPT-I and (2) activation of muscle CPT-Itranscripts by electrical stimulation. (Abstract shortened by UMI.) ^

Mechanism of mRNA stability control mediated by AU -rich element: Characterization of cis-elements and trans-factors

Regulation of cytoplasmic deadenylation, the first step in mRNA turnover, has direct impact on the fate of gene expression. AU-rich elements (AREs) found in the 3′ untranslated regions of many labile mRNAs are the most common RNA-destabilizing elements known in mammalian cells. Based on their sequence features and functional properties, AREs can be divided into three classes. Class I or class III ARE directs synchronous deadenylation, whereas class II ARE directs asynchronous deadenylation with the formation of poly(A)-intermediates. Through systematic mutagenesis study, we found that a cluster of five or six copies of AUUUA motifs forming various degrees of reiteration is the key feature dictating the choice between asynchronous versus synchronous deadenylation. A 20–30 nt AU-rich sequence immediately 5 ′ to this cluster of AUUUA motifs can greatly enhance its destabilizing ability and is an integral part of the AREs. These two features are the defining characteristics of class II AREs. ^ To better understand the decay mechanism of AREs, current methods have several limitations. Taking the advantage of tetracycline-regulated promoter, we developed a new transcriptional pulse strategy, Tet-system. By controlling the time and the amount of Tet addition, a pulse of RNA could be generated. Using this new system, we showed that AREs function in both growth- and density-arrested cells. The new strategy offers for the first time an opportunity to investigate control of mRNA deadenylation and decay kinetics in mammalian cells that exhibit physiologically relevant conditions. ^ As a member of heterogeneous nuclear RNA-binding protein, hnRNP D 0/AUF1 displays specific affinities for ARE sequences in vitro . But its in vivo function in ARE-mediated mRNA decay is unclear. AUF1/hnRNP D0 is composed of at least four isoforms derived by alternative RNA splicing. Each isoform exhibits different affinity for ARE sequence in vitro. Here, we examined in vivo effect of AUF1s/hnRNP D0s on degradation of ARE-containing mRNA. Our results showed that all four isoforms exhibit various RNA stabilizing effects in NIH3T3 cells, which are positively correlated with their binding affinities for ARE sequences. Further experiments indicated that AUF1/hnRNP D0 has a general role in modulating the stability of cytoplasmic mRNAs in mammalian cells. ^