Anush Oganesian

Type IIA Procollagen Amino Propeptide Is Localized in Human Embryonic Tissues

Type II procollagen is synthesized in two forms generated by the alternative splicing of its precursor mRNA. The alternatively spliced domain, exon 2, encodes the 69-amino-acid cysteinerich region of the NH2 propeptide. Studies of mRNA expression have shown that the longer form, designated Type IIA procollagen, is synthesized by chondroprogenitor cells and various noncartilaginous tissues. The shorter form, Type IIB procollagen, is synthesized by differentiated chondrocytes. As the initial step in our investigations of the function of the Type IIA procollagen, the protein domain corresponding to exon 2 was created as a recombinant fusion protein and used to raise antibodies in rabbits. The resulting antiserum was specific for Type IIA procollagen NH2 propeptide as shown by ELISA, Western blotting, and immunofluorescent co-localization with the triple-helical domain of Type II collagen. Type IIA procollagen was identified in tissue culture medium of 54-day human fetal ribs. Confocal microscopy was used to localize the Type IIA NH2 propeptide in Day 50 and 53 human embryos. In the digital rays of the developing hand, where only Type IIA procollagen mRNA was detected, Type IIA procollagen NH2 propeptide was observed in the extracellular matrix. The presence of Type IIA procollagen NH2 propeptide was observed in the cartilage of the developing long bones of the lower arm and vertebral bodies even though these tissues synthesize Type IIB mRNA at this developmental stage. Type IIA procollagen NH2 propeptide was localized in the developing trachea, a cartilage that does not undergo endochondral bone formation. Type IIA NH2 propeptide was also localized in noncartilaginous tissues known to synthesize Type IIA mRNA, such as the intervertebral area, perichondrium, notochordal sheath, and neuroepithelium of the otic vesicle. In most tissues, co-localization with antiserum against the triple-helical domain of Type II collagen was observed. Positive immunoreactivity with the Type IIA NH2 propeptide antiserum indicates, for the first time, that this propeptide is present in the tissue. Co-localization of NH2 propeptide antibodies with the triple-helical domain of the collagen molecule suggests that Type IIA procollagen is intact in the extracellular matrix of these tissues. Taken together, these results strongly suggest that around cells that synthesize Type IIA procollagen mRNA, Type IIA procollagen NH2 propeptide is secreted and deposited into the extracellular matrix. In light of these results, we predict that Type IIA procollagen plays a role in differentiation of tissues that augments its purely architectural function. (J Histochem Cytochem 45:1469–1480, 1997)

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.

The NH2-terminal propeptide of type I procollagen acts intracellularly to modulate cell function

The function of the NH2-terminal propeptide of type I procollagen (N-propeptide) is poorly understood. We now show that a recombinant trimeric N-propeptide interacts with transforming growth factor-β1 and BMP2 and exhibits functional effects in stably transfected cells. The synthesis of N-propeptide by COS-7 cells results in an increase in phosphorylation of Akt and Smad3 and is associated with a marked reduction in type I procollagen synthesis and impairment in adhesion. In C2C12 cells, N-propeptide inhibits the osteoblastic differentiation induced by BMP2. Our data suggest that these effects are mediated by the interaction of N-propeptide with an intracellular receptor in the secretory pathway, because they are not observed when recombinant N-propeptide is added to the culture medium of either COS-7 or C2C12 cells. Both the binding of N-propeptide to cytokines and its functional properties are entirely dependent on the exon 2-encoded globular domain, and a mutation that substitutes a serine for a highly conserved cysteine in exon 2 abolishes its function. Our findings suggest that N-propeptide performs an important feedback regulatory function and provides a rationale for the prominence of a homotrimeric form of type I procollagen (α1 trimer) during vertebrate development.

Constitutive coupling of a naturally occurring human alpha1a-adrenergic receptor genetic variant to EGFR transactivation pathway

We previously identified a naturally occurring human SNP, G247R, in the third intracellular loop of the α1a-adrenergic receptor (α1a-247R) and demonstrated that constitutive expression of α1a-247R results in twofold increased cell proliferation compared with WT. In the present study we elucidate molecular mechanisms and signal transduction pathways responsible for increased cell proliferation unique to α1a-247R, but not α1a-WT, α1b, or α1dAR subtypes. We show that elevated levels of matrix metalloproteinase-7 (MMP7) and a disintegrin and metalloproteinase-12 (ADAM12) in α1a-247R–expressing cells are responsible for EGF receptor (EGFR) transactivation, downstream ERK activation, and increased cell proliferation; this pathway is confirmed using MMP, EGFR, and ERK inhibitors. We demonstrate that EGFR transactivation and downstream ERK activation depends on increased shedding of heparin-binding EGF. Finally, we demonstrate that knockdown of MMP7 or β-arrestin1 by shRNAs results in attenuation of proliferation of cells expressing α1a-247R. Importantly, accelerated cell proliferation triggered by the α1a-247R is serum- and agonist-independent, providing unique evidence for constitutive active coupling to the β-arrestin1/MMP/EGFR transactivation pathway by any G protein-coupled receptor. These findings raise the possibility of a previously unexplored mechanism for sympathetically mediated human hypertension triggered by a naturally occurring human genetic variant.

Alpha1a-adrenoceptor genetic variant induces cardiomyoblast-to-fibroblast-like cell transition via distinct signaling pathways.

The role of naturally occurring human α1a-Adrenergic Receptor (α1aAR) genetic variants associated with cardiovascular disorders is poorly understood. Here, we present the novel findings that expression of human α1aAR-247R (247R) genetic variant in cardiomyoblasts leads to transition of cardiomyoblasts into a fibroblast-like phenotype, evidenced by morphology and distinct de novo expression of characteristic genes. These fibroblast-like cells exhibit constitutive, high proliferative capacity and agonist-induced hypertrophy compared with cells prior to transition. We demonstrate that constitutive, synergistic activation of EGFR, Src and ERK kinases is the potential molecular mechanism of this transition. We also demonstrate that 247R triggers two distinct EGFR transactivation-dependent signaling pathways: 1) constitutive Gq-independent β-arrestin-1/Src/MMP/EGFR/ERK-dependent hyperproliferation and 2) agonist-induced Gq- and EGFR/STAT-dependent hypertrophy. Interestingly, in cardiomyoblasts agonist-independent hyperproliferation is MMP-dependent, but in fibroblast-like cells it is MMP-independent, suggesting that expression of α1aAR genetic variant in cardiomyocytes may trigger extracellular matrix remodeling. Thus, these novel findings demonstrate that EGFR transactivation by α1aAR-247R leads to hyperproliferation, hypertrophy and alterations in cardiomyoblasts, suggesting that these unique genetically-mediated alterations in signaling pathways and cellular function may lead to myocardial fibrosis. Such extracellular matrix remodeling may contribute to the genesis of arrhythmias in certain types of heart failure.

Alpha1a-Adrenoceptor Genetic Variant Triggers Vascular Smooth Muscle Cell Hyperproliferation and Agonist Induced Hypertrophy via EGFR Transactivation Pathway.

α1a Adrenergic receptors (α1aARs) are the predominant AR subtype in human vascular smooth muscle cells (SMCs). α1aARs in resistance vessels are crucial in the control of blood pressure, yet the impact of naturally occurring human α1aAR genetic variants in cardiovascular disorders remains poorly understood. To this end, we present novel findings demonstrating that 3D cultures of vascular SMCs expressing human α1aAR-247R (247R) genetic variant demonstrate significantly increased SMC contractility compared with cells expressing the α1aAR-WT (WT) receptor. Stable expression of 247R genetic variant also triggers MMP/EGFR-transactivation dependent serum- and agonist-independent (constitutive) hyperproliferation and agonist-dependent hypertrophy of SMCs. Agonist stimulation reduces contractility Using pathway-specific inhibitors we determined that the observed hyperproliferation of 247R-expressing cells is triggered via β-arrestin1/Src/MMP-2/EGFR/ERK-dependent mechanism. MMP-2-specific siRNA inhibited 247R-triggered hyperproliferation indicating MMP-2 involvement in 247R-triggered hyperproliferation in SMCs. β-arrestin1-specific shRNA also inhibited 247R-triggered hyperproliferation but did not affect hypertrophy in 247R-expressing SMCs, indicating that agonist-dependent hypertrophy is independent of β-arrestin1. Our data reveal that in different cardiovascular cells the same human receptor genetic variant can activate alternative modulators of the same signaling pathway. Thus, our findings in SMCs demonstrate that depending on the type of cells expressing the same receptor (or receptor variant), different target-specific inhibitors could be used to modulate aberrant hyperproliferative or hypertrophic pathways in order to restore normal phenotype.