Scott A. Coats

Matrix Metalloproteinase-9 Overexpression Enhances Vascular Smooth Muscle Cell Migration and Alters Remodeling in the Injured Rat Carotid Artery

Matrix metalloproteinase-9 (MMP-9) has been implicated in the pathogenesis of atherosclerosis as well as intimal hyperplasia after vascular injury. We used Fischer rat smooth muscle cells (SMCs) overexpressing MMP-9 to determine the role of MMP-9 in migration and proliferation as well as in vessel remodeling after balloon denudation. Fischer rat SMCs were stably transfected with a cDNA for rat MMP-9 under the control of a tetracycline-regulatable promoter. In this system, MMP-9 was overexpressed in the absence, but not in the presence, of tetracycline. In vitro SMC migration was determined using a collagen invasion assay as well as a Boyden chamber assay. In vivo migration was determined by measuring the invasion into the medial and intimal layers of transduced SMCs seeded on the outside of the artery. Transduced SMCs were also seeded on the luminal surface, and the effect of local MMP-9 overexpression on vascular structure was measured morphometrically at intervals up to 28 days. MMP-9 overexpression enhanced SMC migration in both the collagen invasion assay and Boyden chamber in vitro, increased SMC migration into an arterial matrix in vivo, and altered vessel remodeling by increasing the vessel circumference, thinning the vessel wall and decreasing intimal matrix content. These results demonstrate that MMP-9 enhances vascular SMC migration in vitro and in vivo and alters postinjury vascular remodeling.

Fas/FADD-mediated activation of a specific program of inflammatory gene expression in vascular smooth muscle cells.

Apoptosis of smooth muscle cells is a common feature of vascular lesions but its pathophysiological significance is not known. We demonstrate that signals initiated by regulated Fas-associated death domain protein overexpression in rat vascular smooth muscle cells in the carotid artery induce expression of monocyte-chemoattractant protein-1 and interleukin-8, and cause massive immigration of macrophages in vivo. These chemokines, and a specific set of other pro-inflammatory genes, are also upregulated in human vascular smooth muscle cells during Fas-induced apoptosis, in part through a process that requires interleukin-1α activation. Induction of a pro-inflammatory program by apoptotic vascular smooth muscle cells may thus contribute to the pathogenesis of vascular disease.

Germline ETV6 mutations in familial thrombocytopenia and hematologic malignancy

We report germline missense mutations in ETV6 segregating with the dominant transmission of thrombocytopenia and hematologic malignancy in three unrelated kindreds, defining a new hereditary syndrome featuring thrombocytopenia with susceptibility to diverse hematologic neoplasms. Two variants, p.Arg369Gln and p.Arg399Cys, reside in the highly conserved ETS DNA-binding domain. The third variant, p.Pro214Leu, lies within the internal linker domain, which regulates DNA binding. These three amino acid sites correspond to hotspots for recurrent somatic mutation in malignancies. Functional studies show that the mutations abrogate DNA binding, alter subcellular localization, decrease transcriptional repression in a dominant-negative fashion and impair hematopoiesis. These familial genetic studies identify a central role for ETV6 in hematopoiesis and malignant transformation. The identification of germline predisposition to cytopenias and cancer informs the diagnosis and medical management of at-risk individuals.

Mitotic spindle destabilization and genomic instability in Shwachman-Diamond syndrome

Deficiencies in the SBDS gene result in Shwachman-Diamond syndrome (SDS), an inherited bone marrow failure syndrome associated with leukemia predisposition. SBDS encodes a highly conserved protein previously implicated in ribosome biogenesis. Using human primary bone marrow stromal cells (BMSCs), lymphoblasts, and skin fibroblasts, we show that SBDS stabilized the mitotic spindle to prevent genomic instability. SBDS colocalized with the mitotic spindle in control primary BMSCs, lymphoblasts, and skin fibroblasts and bound to purified microtubules. Recombinant SBDS protein stabilized microtubules in vitro. We observed that primary BMSCs and lymphoblasts from SDS patients exhibited an increased incidence of abnormal mitoses. Similarly, depletion of SBDS by siRNA in human skin fibroblasts resulted in increased mitotic abnormalities and aneuploidy that accumulated over time. Treatment of primary BMSCs and lymphoblasts from SDS patients with nocodazole, a microtubule destabilizing agent, led to increased mitotic arrest and apoptosis, consistent with spindle destabilization. Conversely, SDS patient cells were resistant to taxol, a microtubule stabilizing agent. These findings suggest that spindle instability in SDS contributes to bone marrow failure and leukemogenesis.

Vasodilator-Stimulated Phosphoprotein Regulates Proliferation and Growth Inhibition by Nitric Oxide in Vascular Smooth Muscle Cells

Objective—Vasodilator-stimulated phosphoprotein (VASP) was identified as a substrate for cGMP-dependent protein kinase (PKG) and cAMP-dependent protein kinase (PKA). It is preferentially phosphorylated at serine239 by PKG, whereas serine157 is a preferred phosphorylation site for PKA. In addition, serine157 is phosphorylated by PKC in response to serum. We have investigated the effects of VASP and VASP phosphorylation at serine157 and serine239 on smooth muscle cell (SMC) proliferation and nitric oxide (NO)-mediated growth inhibition. Methods and Results—Aortic SMCs derived from VASP-deficient mice were transduced with retroviral vectors encoding either wild-type VASP or VASP mutants (S157A-VASP and S239A-VASP), in which serine157 and serine239, respectively, were replaced by a nonphosphorylatable amino acid, alanine. Expression of wt-VASP and S239A-VASP significantly increased proliferation, whereas expression of S157A-VASP was inhibitory. Expression of S239A-VASP rendered SMCs less sensitive to growth inhibition by the NO donor, S-nitroso-n-acetylpenicillamine, when compared with cells expressing wt-VASP. Similar effects were observed in cultured rat SMCs in which wt-VASP, S157A-VASP, and S239A-VASP were expressed. Conclusions—Our data suggest that VASP phosphorylation at serine157 is required for the growth-stimulatory effect of VASP in SMCs, whereas VASP phosphorylation at serine239 is involved in the growth inhibitory effects of NO on SMCs.

Impaired ribosomal subunit association in Shwachman-Diamond syndrome

Shwachman-Diamond syndrome (SDS) is an autosomal-recessive marrow failure syndrome with a predisposition to leukemia. SDS patients harbor biallelic mutations in the SBDS gene, resulting in low levels of SBDS protein. Data from nonhuman models demonstrate that the SBDS protein facilitates the release of eIF6, a factor that prevents ribosome joining. The complete abrogation of Sbds expression in these models results in severe cellular and lethal physiologic abnormalities that differ from the human disease phenotype. Because human SDS cells are characterized by partial rather than complete loss of SBDS expression, we interrogated SDS patient cells for defects in ribosomal assembly. SDS patient cells exhibit altered ribosomal profiles and impaired association of the 40S and 60S subunits. Introduction of a wild-type SBDS cDNA into SDS patient cells corrected the ribosomal association defect, while patient-derived SBDS point mutants only partially improved subunit association. Knockdown of eIF6 expression improved ribosomal subunit association but did not correct the hematopoietic defect of SBDS-deficient cells. In summary, we demonstrate an SBDS-dependent ribosome maturation defect in SDS patient cells. The role of ribosomal subunit joining in marrow failure warrants further investigation.

The Neurosecretory Vesicle Protein Phogrin Functions as a Phosphatidylinositol Phosphatase to Regulate Insulin Secretion

Phogrin is a transmembrane protein expressed in cells with stimulus-coupled peptide hormone secretion, including pancreatic beta cells, in which it is localized to the membrane of insulin-containing dense-core vesicles. By sequence, phogrin is a member of the family of receptor-like protein-tyrosine phosphatases, but it contains substitutions in conserved catalytic sequences, and no significant enzymatic activity for phogrin has ever been reported. We report here that phogrin is able to dephosphorylate specific inositol phospholipids, including phosphatidylinositol (PI) 3-phosphate and PI 4,5-diphosphate but not PI 3,4,5-trisphosphate. The phosphatidylinositol phosphatase (PIPase) activity of phogrin was measurable but low when evaluated by the ability of a catalytic domain fusion protein to hydrolyze soluble short-chain phosphatidylinositol phospholipids. Unlike most PIPases, which are cytoplasmic proteins that associate with membranes, mature phogrin is a transmembrane protein. When the transmembrane form of phogrin was overexpressed in mammalian cells, it reduced plasma membrane phosphatidylinositol 4,5-disphosphate levels in a dose-dependent manner. When purified and assayed in vitro, the transmembrane form had a specific activity of 142 mol/min/mol, 75-fold more active than the catalytic domain fusion protein and comparable with the specific activities of the other PIPases. The PIPase activity of phogrin depended on the catalytic site cysteine and correlated with effects on glucose-stimulated insulin secretion. We propose that phogrin functions as a phosphatidylinositol phosphatase that contributes to maintaining subcellular differences in levels of PIP that are important for regulating stimulus-coupled exocytosis of insulin.

PTPRQ is a novel phosphatidylinositol phosphatase that can be expressed as a cytoplasmic protein or as a subcellularly localized receptor-like protein

PTPRQ (rPTP-GMC1) is a member of the type III receptor-like protein tyrosine phosphatase family. PTPRQ has very low activity against phosphotyrosine but is active against phosphatidylinositol phosphates that are involved in regulation of survival, proliferation, and subcellular architecture. Here, we report that PTPRQ can be expressed as a cytosolic or a receptor-like protein and that the form, subcellular localization, and cell types in which it is expressed are regulated by alternative promoter use and by alternative splicing. The first promoter drives expression of transcripts encoding a transmembrane protein in human podocytes and lung. PTPRQ protein is localized to the basal membrane of human podocytes, beginning when podocyte progenitors can first be identified in the embryonic kidney. A second promoter drives expression of a transcript that can encode a cytoplasmic protein containing the catalytic site. This is the major PTPRQ transcript in rat mesangial cells and human testis and is upregulated in mesangial cells in a rat model of mesangial proliferative glomerulonephritis. Differential regulation of expression of the transmembrane vs cytosolic forms, in different cell types during development or response to injury, may be a mechanism through which PTPRQ, with its activities against membrane phospholipids and against phosphotyrosine, can target specific substrates under different conditions.