Francesco Ramirez

Cell signaling events: a view from the matrix.

Cellular activities are primarily initiated, modulated and sustained by multifunctional molecules (cytokines and growth factors) that are secreted into the extracellular space and that signal through membrane-bound, high-affinity receptors. In contrast to the fairly well understood mechanisms that mediate the specificity of signal transduction within the confined and compartmentalized environment of the cell, significantly less is known about the mechanisms that regulate the availability of signaling molecules in the extracellular milieu. Recent findings have implicated the participation of extracellular protein macroaggregates in signaling events controlling patterning and morphogenesis. The results suggest a functional coupling between the tissue-specific organization of collagenous and elastic macroaggregates and their ability to perform instructive as well as structural functions. These observations open the way to a novel understanding in these poorly understood and critically important areas of cell and developmental biology.

Collagen XXIV, a vertebrate fibrillar collagen with structural features of invertebrate collagens: Selective expression in developing cornea and bone

Tissue-specific assembly of fibers composed of the major collagen types I and II depends in part on the formation of heterotypic fibrils, using the quantitatively minor collagens V and XI. Here we report the identification of a new fibrillar-like collagen chain that is related to the fibrillar α1(V), α1(XI), and α2(XI) collagen polypeptides and which is coexpressed with type I collagen in the developing bone and eye. The new collagen was designated the α1(XXIV) chain and consists of a long triple helical domain flanked by typical propeptide-like sequences. The carboxyl propeptide is classic, with 8 conserved cysteine residues. The amino-terminal peptide contains a thrombospodin-N-terminal-like (TSP) motif and a highly charged segment interspersed with several tyrosine residues, like the fibril diameter-regulating collagen chains α1(V) and α1(XI). However, a short imperfection in the triple helix makes α1(XXIV) unique from other chains of the vertebrate fibrillar collagen family. The triple helical interruption and additional select features in both terminal peptides are common to the fibrillar chains of invertebrate organisms. Based on these data, we propose that collagen XXIV is an ancient molecule that may contribute to the regulation of type I collagen fibrillogenesis at specific anatomical locations during fetal development.

Early response of α2(I) collagen to acetaldehyde in human hepatic stellate cells is TGF-β 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 H2O2 and thus establish a direct connection between oxidative stress and collagen upregulation. We localized to the −378 to −183 region of the α2(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 β (TGF‐β)1–responsive element, and both acetaldehyde and this cytokine induce their effects through H2O2, 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‐β1. These include significant time‐course differences in the expression of COL1A2 and TGF‐β1 mRNAs and inability of neutralizing antibodies to TGF‐β1 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‐β1 was PI3K independent. In conclusion, acetaldehyde‐dependent mechanisms involved in COL1A2 upregulation are similar, but not identical, to those of TGF‐β1. We suggest that early acetaldehyde‐dependent events induce the late expression of TGF‐β1 and create an H2O2‐dependent autocrine loop that may sustain and amplify the fibrogenic response of this alcohol metabolite. (HEPATOLOGY 2005;42:343–352.)

Cooperativity between Far Upstream Enhancer and Proximal Promoter Elements of the Human α2(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 α2(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.

Magnetic resonance microscopy quantifies the disease progression in Marfan syndrome mice

To use noninvasive magnetic resonance microscopy (MRM) to examine aneurysmal disease in the mouse model of Marfan syndrome (MFS).