Biomedical subjects

Francesco Ramirez

Publications and source records attributed to Francesco Ramirez.

At least 19 recordsLinked to original sources

PubMed2007

Symposium on the musculoskeletal aspects of Marfan syndrome: meeting report and state of the science.

The National Marfan Foundation sponsored a symposium in August 2005 to review recent progress in the area of Marfan-related musculoskeletal research. Orthopaedic surgeons, molecular geneticists, medical geneticists, and pain specialists met to review a variety of topics. This report reviews and summarizes the proceedings of the symposium, with emphasis on future directions for study that were identified in the course of the meeting. Areas covered include clinical detection, diagnosis, growth, spine deformity, molecular mechanisms, dural ectasia, protrusio acetabuli, and pain in Marfan syndrome.

Acetabulum↗

PubMed2006

The first intron of the human alpha2(I) collagen gene (COL1A2) contains a novel interferon-gamma responsive element.

Cell transfection assays have shown that several transcription factors can mediate interferon-gamma (INF-gamma) inhibition of the human alpha2(I) collagen gene (COL1A2) by binding distinct cis-acting elements in the proximal promoter. Recent transgenic work, on the other hand, has identified a strong repressor in the first intron of COL1A2 that includes a binding site for interferon regulatory factors (IRFs). Here we present evidence from cell transfection experiments indicating that this IRF-binding site (IF3) is a novel target of the pathways elicited by INF-gamma to blunt transcription from the COL1A2 promoter. First, we showed that INF-gamma stimulates the production of IRF-1 transcripts, as well as the formation of an IRF-1 containing complex at the FI3 element. Second, we demonstrated that IRF-1 over-expression inhibits COL1A2 promoter activity specifically through the action of FI3, in addition to decreasing the steady-state levels of the endogenous COL1A2 mRNA. Third, we documented that INF-gamma treatment of cultured fibroblasts increases binding of IRF-1 to FI3 in the endogenous COL1A2 gene. Together our findings further extend the list of transcription factors involved in INF-gamma inhibition of COL1A2 gene expression.

Animals↗

PubMed2006

Identification of genes regulated by transcription factor KLF7 in differentiating olfactory sensory neurons.

Gene targeting in mice has recently demonstrated that transcription factor KLF7 plays a critical role in neurite outgrowth and neuronal survival. Here we extended this genetic evidence by establishing the transcriptional profile of differentiating olfactory sensory neurons (OSNs) in Klf7(-/-) mice, and by identifying relevant genes that are directly regulated by KLF7. Functional clustering of DNA microarray data revealed that loss of KLF7 affects primarily the activity of genes involved in OSN differentiation, axonal growth, cytoskeletal dynamics, cell adhesion and synaptogenesis. Cell transfection experiments, on the other hand, demonstrated that the promoters of the genes encoding the OSN-specific OMP and the adhesion molecule L1 are both activated by KLF7 binding to CACCC motifs. Collectively, these results advance knowledge of transcriptional regulation of olfactory neurogenesis and KLF7 action.

Animals↗

PubMed2006

Multiple pathways regulate intracellular shuttling of MoKA, a co-activator of transcription factor KLF7.

MoKA is a novel F-box containing protein that interacts with and stimulates the activity of transcription factor KLF7, a regulator of neuronal differentiation. MoKA accumulates throughout the cell and predominantly in the cytosol, consistent with the presence of several putative nuclear localization and export signals (NLSs and NESs). The present study was designed to refine the identity and location of the sequences responsible for MoKA intracellular shuttling and transcriptional activity. Forced expression of fusion proteins in mammalian cells demonstrated that only one of three putative NLSs potentially recognized by karyopherin receptors is involved in nuclear localization of MoKA. By contrast, three distinct sequences were found to participate in mediating cytoplasmic accumulation. One of them is structurally and functionally related to the leucine-rich export signal that interacts with the exportin 1 (CRM1) receptor. The other two export signals instead display either a novel leucine-rich sequence or an undefined peptide motif, and both appear to act through CRM1-independent pathways. Finally, transcriptional analyses using the chimeric GAL4 system mapped the major activation domain of MoKA to a highly acidic sequence that resides between the NLS and NES clusters.

Animals↗

PubMed2006

Mice without transcription factor KLF7 provide new insight into olfactory bulb development.

Recent genetic studies have excluded that peripheral innervation plays a substantial role in the initial outgrowth of the olfactory bulb. Mice without Kruppel-like factor 7 activity die at birth and display hypoplastic olfactory bulbs which lack peripheral innervation. Here, we report that incomplete penetrance of the mutation is responsible for partial bulb innervation in a small fraction of Klf7 null mice. Analysis of the partially innervated bulbs of mutant embryos, newborns and adult mice revealed an obligatory correlation with local restoration of laminar architecture, neuronal cell differentiation and neuronal activity. The degree of normal OB maturation in Klf7-/- OBs was proportional to the degree of peripheral innervation. These findings therefore indicate that peripheral innervation contributes to bulb maturation late in development by promoting cell morphogenesis and differentiation.

Animals↗

PubMed2006

Transcriptional regulation of the human alpha2(I) collagen gene (COL1A2), an informative model system to study fibrotic diseases.

During the past two decades, the human pro-alpha2(I) collagen gene (COL1A2) has emerged as an informative model in which to study the general principles that govern the transcriptional control of extracellular matrix deposition in normal and fibrotic conditions. Multiple studies have in fact delineated the genomic regions, cis-acting elements and trans-acting factors implicated in constitutive, cytokine-modulated and tissue-specific expression of COL1A2. These functional components are integrated into a regulatory network that consists of the proximal promoter, far-upstream enhancer and downstream repressor, and which operates according to two mechanisms. The first mechanism is one in which combinatorial interactions among promoter-bound proteins determine transcriptional outcome in different cellular and experimental contexts. The other mechanism is one whereby cooperative assembly of protein complexes at distantly located DNA elements directs spatiotemporal specificity. These transcriptional studies have also an additional value in translational research, in that they are providing the conceptual means to develop new animal models of and therapeutic strategies for fibrotic diseases.

Animals↗

PubMed2006

Losartan, an AT1 antagonist, prevents aortic aneurysm in a mouse model of Marfan syndrome.

Aortic aneurysm and dissection are manifestations of Marfan syndrome (MFS), a disorder caused by mutations in the gene that encodes fibrillin-1. Selected manifestations of MFS reflect excessive signaling by the transforming growth factor-beta (TGF-beta) family of cytokines. We show that aortic aneurysm in a mouse model of MFS is associated with increased TGF-beta signaling and can be prevented by TGF-beta antagonists such as TGF-beta-neutralizing antibody or the angiotensin II type 1 receptor (AT1) blocker, losartan. AT1 antagonism also partially reversed noncardiovascular manifestations of MFS, including impaired alveolar septation. These data suggest that losartan, a drug already in clinical use for hypertension, merits investigation as a therapeutic strategy for patients with MFS and has the potential to prevent the major life-threatening manifestation of this disorder.

Adrenergic beta-Antagonists↗

PubMed2005

Fibrillins 1 and 2 perform partially overlapping functions during aortic development.

Fibrillin-rich microfibrils are extracellular assemblies that impart structural properties to the connective tissue. To elucidate the contribution of fibrillin-rich microfibrils to organogenesis, we have examined the vascular phenotype of a newly created strain of mice that completely lacks fibrillin-1 and the consequences of combined deficiency of fibrillins 1 and 2 on tissue formation. The results demonstrated that fibrillins 1 and 2 perform partially overlapping functions during aortic development. Fbn1-/- mice died soon after birth from ruptured aortic aneurysm, impaired pulmonary function, and/or diaphragmatic collapse. Analysis of the neonatal Fbn1-/- aorta documented a disorganized and poorly developed medial layer but normal levels of elastin cross-links. Transcriptional profiling revealed that aneurysm progression in Fbn1 null mice is accompanied by unproductive up-regulation of gene products normally involved in tissue repair and vascular integrity, such as plasminogen activator inhibitor-1, activin A, and cysteine-rich angiogenic protein 61. In contrast to Fbn1-/- mice, Fbn2 null mice had a well developed and morphologically normal aortic wall. However, virtually all Fbn1-/-;Fbn2-/- embryos and about half of the Fbn1+/-;Fbn2-/- embryos died in utero and displayed a significantly more severe vascular phenotype than Fbn1-/- mice. Consistent with a specialized function of fibrillin-2, electron microscopy visualized ultrastructurally different microfibrils in Fbn1 null compared with control cell cultures. Collectively, these data demonstrate that involvement of fibrillin-2 in the initial assembly of the aortic matrix overlaps in part with fibrillin-1 and that continued fibrillin-1 deposition is absolutely required for the maturation and function of the vessel during neonatal life.

Activins↗

PubMed2005

CREB-AP1 protein complexes regulate transcription of the collagen XXIV gene (Col24a1) in osteoblasts.

Collagen XXIV is a newly discovered and poorly characterized member of the fibril-forming family of collagen molecules, which displays unique structural features of invertebrate fibrillar collagens and is expressed predominantly in bone tissue. Here we report the characterization of the proximal promoter of the mouse gene (Col24a1) and its regulation in osteoblastic cells. Using well characterized murine models of osteoblast differentiation, we found that the Col24a1 gene is activated sometime before onset of the late differentiation marker osteocalcin. Additional analyses revealed that Col24a1 produces equal amounts of two alternatively spliced products with different 5'-untranslated sequences that originate from distinct transcriptional start sites. Cell transfection experiments in combination with DNA binding assays demonstrated that Col24a1 promoter activity in ROS17/2.8 osteosarcoma cells is under the control of an upstream cis-acting element, which is shared by both transcripts and is recognized by specific combinations of c-Jun, CREB1, ATF1, and ATF2 dimers. Consistent with these results, overexpression of c-Jun, ATF1, ATF2, or CREB1 in transiently transfected osteoblastic cells stimulated transcription from reporter gene constructs driven by the Col24a1 promoter to different degrees. Moreover, chromatin immunoprecipitation experiments showed that these nuclear factors bind the same upstream sequence of the endogenous Col24a1 gene. Collectively these data provide new information about transcriptional control of collagen fibrillogenesis, in addition to implicating for the first time CREB-AP1 protein complexes in the regulation of collagen gene expression in osteoblasts.

Alternative Splicing↗

PubMed2005

Recent progress towards a molecular understanding of Marfan syndrome.

Marfan syndrome (MFS) is a systemic disorder of the connective tissue that is inherited as an autosomal dominant trait and which displays variable manifestations in the ocular, skeletal, and cardiovascular systems. These pleiotropic manifestations are accounted for by mutations in fibrillin-1, the building block of extracellular microfibrils. During the past 10 years, we have witnessed significant progress in delineating the pathological events responsible for the manifestations of MFS. Much of this progress has been based on the creation and analysis of fibrillin-1 mutant mouse lines that faithfully recapitulate the spectrum of clinical severity of MFS. These studies have established the critical contribution of fibrillin-1 deficiency to disease progression through altered cell-matrix interactions and dysregulated TGF-beta signaling. As a result, our definition of MFS as the prototypical structural disorder of the connective tissue has changed to that of a developmental abnormality with broad and complex effects on the morphogenesis and function of multiple organ systems. Importantly, new biological targets have emerged that may yield exciting new opportunities for the development of productive treatment strategies in MFS.

Animals↗

PubMed2005

Developmental regulation of yolk sac hematopoiesis by Kruppel-like factor 6.

Krüppel-like factor 6 (KLF6) is a member of a growing family of transcription factors that share a common 3 C2H2 zinc finger DNA binding domain and have broad activity in regulating proliferation and development. We have previously established that Klf6 is expressed in neuronal tissue, hindgut, heart, lung, kidney, and limb buds during midgestation. To explore the potential role of Klf6 in mouse development, we analyzed Klf6-/- mice and found that the homozygous mutation is embryonic lethal by embryonic day (E) 12.5 and associated with markedly reduced hematopoiesis and poorly organized yolk sac vascularization. Additionally, mRNA levels of Scl and Gata1 were reduced by approximately 80% in Klf6-/- yolk sacs. To further analyze this phenotype, we generated Klf6-/- embryonic stem (ES) cells by homologous recombination, and compared their capacity to differentiate into the hematopoietic lineage with that of either Klf6+/- or Klf6+/+ ES cells. Consistent with the phenotype in the early embryo, Klf6-/- ES cells displayed significant hematopoietic defects following differentiation into EBs. Prolongation of epiblast-like cells and delays in mesoderm induction were also observed in the Klf6-/- EBs, associated with delayed expression of Brachyury, Klf1, and Gata1. Forced expression of KLF6 using a tet-inducible system enhanced the hematopoietic potential of wild-type EBs. Collectively, these findings implicate Klf6 in ES-cell differentiation and hematopoiesis.

Animals↗

PubMed2005

Identification of a repressor in the first intron of the human alpha2(I) collagen gene (COL1A2).

The human and mouse genes that code for the alpha2 chain of collagen I (COL1A2 and Col1a2, respectively) share a common chromatin structure and nearly identical proximal promoter and far upstream enhancer sequences. Despite these homologies, species-specific differences have been reported regarding the function of individual cis-acting elements, such as the first intron sequence. In the present study, we have investigated the transcriptional contribution of the unique open chromatin site in the first intron of COL1A2 using a transgenic mouse model. DNase I footprinting identified a cluster of three distinct areas of nuclease protection (FI1-3) that span from nucleotides +647 to +760, relative to the transcription start site, and which contain consensus sequences for GATA and interferon regulatory factor (IRF) transcription factors. Gel mobility shift and chromatin immunoprecipitation assays corroborated this last finding by documenting binding of GATA-4 and IRF-1 and IRF-2 to the first intron sequence. Moreover, a short sequence encompassing the three footprints was found to inhibit expression of transgenic constructs containing the COL1A2 proximal promoter and far upstream enhancer in a position-independent manner. Mutations inserted into each of the footprints restored transgenic expression to different extents. These results therefore indicated that the unique open chromatin site of COL1A2 corresponds to a repressor, the activity of which seems to be mediated by the concerted action of GATA and IRF proteins. More generally, the study reiterated the existence of species-specific difference in the regulatory networks of the mammalian alpha2(I) collagen coding genes.

Animals↗

PubMed2005

The zinc finger transcription factor Klf7 is required for TrkA gene expression and development of nociceptive sensory neurons.

TrkA, the high affinity receptor for nerve growth factor (NGF), is essential for the development of nociceptive sensory and sympathetic neurons. The zinc finger transcription factor Klf7 interacts with an important cis element of the TrkA minimal enhancer and is coexpressed with TrkA in these neurons. We show that Klf7 binds to the endogenous TrkA minimal enhancer and can activate transcription from the TrkA minimal enhancer in a sequence-dependent manner. In Klf7(-/-) newborn mice, we find a significant reduction in sensory neurons due to increased apoptosis. The neuronal loss is restricted to nociceptive neurons that normally depend on TrkA for neurotrophic support, while other populations of somatosensory neurons appear normal. The reduction of TrkA expression in sensory neurons is a direct effect of Klf7 gene ablation, rather than a secondary effect of cell death. As a result, Klf7(-/-) mice have deficient response to noxious stimuli. Finally, removal of one TrkA allele exacerbates the loss of TrkA(+) neurons in Klf7(-/-) mice. Thus, Klf7 specifically regulates TrkA gene expression and is required for the development of a subset of nociceptive sensory neurons.

Alleles↗

PubMed2005

Early response of alpha2(I) collagen to acetaldehyde in human hepatic stellate cells is TGF-beta independent.

Acetaldehyde is fibrogenic and induces the expression of type I collagen genes in hepatic stellate cells. Some of these acetaldehyde-dependent events are mediated by H(2)O(2) and thus establish a direct connection between oxidative stress and collagen upregulation. We localized to the -378 to -183 region of the alpha2(I) collagen (COL1A2) promoter an acetaldehyde-responsive element (AcRE) functional in human hepatic stellate cells (HHSCs) and investigated molecular mechanisms whereby acetaldehyde stimulates and modulates its transcriptional activity. Because the AcRE co-localized with a previously described transforming growth factor beta (TGF-beta)1-responsive element, and both acetaldehyde and this cytokine induce their effects through H(2)O(2), we investigated whether all fibrogenic actions of acetaldehyde were mediated by this cytokine. Here we show that acetaldehyde-induced COL1A2 upregulation in HHSCs recognizes two distinct but overlapping early and late stages that last from 1 to 6 hours and from 6 to 24 hours, respectively. We present several lines of evidence to show that early acetaldehyde-mediated events are independent of TGF-beta1. These include significant time-course differences in the expression of COL1A2 and TGF-beta1 mRNAs and inability of neutralizing antibodies to TGF-beta1 to inhibit acetaldehyde-dependent collagen gene transcription and Smad 3 phosphorylation. We also show that although acetaldehyde-dependent upregulation of collagen was PI3K dependent, that of TGF-beta1 was PI3K independent. In conclusion, acetaldehyde-dependent mechanisms involved in COL1A2 upregulation are similar, but not identical, to those of TGF-beta1. We suggest that early acetaldehyde-dependent events induce the late expression of TGF-beta1 and create an H(2)O(2)-dependent autocrine loop that may sustain and amplify the fibrogenic response of this alcohol metabolite.

Acetaldehyde↗

PubMed2005

GATA-4 binds to an upstream element of the human alpha2(I) collagen gene (COL1A2) and inhibits transcription in fibroblasts.

Chromatin analyses have identified two DNase I hypersensitive sites (HS1 and HS2) at comparable distances (-130 bp and -2.3 kb) from the transcription start site of the human and mouse alpha2I collagen gene. Whereas the DNA region encompassing HS1 has been extensively characterized using protein binding and functional assays, nothing is yet known about the contribution of the HS2 sequence to alpha2I collagen gene transcription. Here we report that the HS2 sequence of the human alpha2I collagen (COL1A2) gene is a binding site for a transcriptional repressor in fibroblasts. DNase I footprinting identified a single site of nuclease protection around HS2, which corresponds to a sequence potentially capable of forming a 13 bp long hairpin structure with a 4 bp interruption in the middle. Gel mobility shift assays revealed that two GATA consensus sequences embedded within the hairpin are specifically bound by GATA-4 in fibroblasts. They also showed that formation of the HS2 protein complex requires the integrity of the whole hairpin sequence. Transient transfections of luciferase reporter gene constructs in fibroblasts correlated the HS2 element with transcriptional repression of the -2.3 kb promoter sequence. This last observation was further corroborated by showing that forced overexpression of GATA-4 in cultured fibroblasts leads to decreased transcription from the co-transfected -2.3 kb promoter/reporter construct, as well as reduced expression of the endogenous collagen gene. Finally, a chromatin immunoprecipitation assay documented GATA-4 ability to bind to the HS2 element in vivo. These results are therefore the first to implicate GATA-4 in regulating constitutive COL1A2 gene expression in fibroblasts.

Animals↗

PubMed2005

Transcription factor KLF7 is important for neuronal morphogenesis in selected regions of the nervous system.

The Krüppel-like transcription factors (KLFs) are important regulators of cell proliferation and differentiation in several different organ systems. The mouse Klf7 gene is strongly active in postmitotic neuroblasts of the developing nervous system, and the corresponding protein stimulates transcription of the cyclin-dependent kinase inhibitor p21waf/cip gene. Here we report that loss of KLF7 activity in mice leads to neonatal lethality and a complex phenotype which is associated with deficits in neurite outgrowth and axonal misprojection at selected anatomical locations of the nervous system. Affected axon pathways include those of the olfactory and visual systems, the cerebral cortex, and the hippocampus. In situ hybridizations and immunoblots correlated loss of KLF7 activity in the olfactory epithelium with significant downregulation of the p21waf/cip and p27kip1 genes. Cotransfection experiments extended the last finding by documenting KLF7's ability to transactivate a reporter gene construct driven by the proximal promoter of p27kip1. Consistent with emerging evidence for a role of Cip/Kip proteins in cytoskeletal dynamics, we also documented p21waf/cip and p27kip1 accumulation in the cytoplasm of differentiating olfactory sensory neurons. KLF7 activity might therefore control neuronal morphogenesis in part by optimizing the levels of molecules that promote axon outgrowth.

Animals↗

PubMed2004

Cooperativity between far upstream enhancer and proximal promoter elements of the human {alpha}2(I) collagen (COL1A2) gene instructs tissue specificity in transgenic mice.

Interaction between the proximal (-378) promoter and the far upstream (-20 kb) enhancer is essential for tissue-specific expression of the human alpha2(I) collagen gene (COL1A2) in transgenic mice. Previous in vitro studies have shown that three Sp1 binding sites (around -300) are part of a cytokine-responsive element and that two TC-rich boxes (around -160 and -125) and a CBF/NFY consensus sequence (around -80) confer optimal promoter activity by interacting among themselves and with the upstream Sp1 sites. Here we report that mutations of the Sp1 binding sites, TC-rich boxes or CBF/NFY consensus sequence lead to reduced transgene activity, thus underscoring the functional interdependence of the proximal promoter elements. Loss of the Sp1 binding sites was associated with loss of transgene expression in osteoblasts, whereas elimination of the CBF/NFY binding site (alone or in combination with the TC-rich boxes) was correlated with a lack of activity in the ventral fascia and head dermis and musculature. Additionally, transgene expression in skin fascia fibroblasts depended on the integrity of the Sp1 binding sites and TC-rich boxes, and on their physical configuration. Evidence is also presented suggesting cooperativity between cis-acting elements of the far upstream enhancer and proximal promoter in assembling tissue-specific protein complexes. This study thus reiterates the complex and highly combinatorial nature of the regulatory network governing COL1A2 transcription in vivo.

Animals↗

PubMed2004

Fibrillin microfibrils: multipurpose extracellular networks in organismal physiology.

Organismal physiology depends significantly on the proper assembly of extracellular matrix (ECM) macroaggregates that impart structural integrity to the connective tissue. Recent genetic studies in mice have unraveled unsuspected new functions of architectural matrix components in regulating signaling events that modulate patterning, morphogenesis, and growth of several organ systems. As a result, a new paradigm has emerged whereby tissue-specific organization of the ECM dictates not only the physical properties of the connective tissue, but also the ability of the matrix to direct a broad spectrum of cellular activities through the regulation of growth factor signaling. These observations pave the way to novel therapeutic approaches aimed at counteracting the deleterious consequences of perturbations of connective tissue homeostasis.

Animals↗