Sandra K. Masur

Tumor-specific cell surface expression of the -KDEL containing endoplasmic reticular heat shock protein gp96

Heat shock protein (HSP) gp96/grp94 contains a signal peptide at the amino terminus and a ‐KDEL sequence at the carboxy terminus and is a major component of the lumen of the mammalian endoplasmic reticulum (ER). We show, by a number of immunolocalization methods using light and electron microscopy, that a significant proportion of intact gp96 molecules is also expressed on the cell surface. Surface gp96 molecules truly represent surface expression and do not result from adventitious deposition of gp96 released by dead cells on to the live cells in culture. Cell surface expression of gp96 is enhanced by heat shock and exposure to reducing agents. Gp96 molecules are not released from plasma membranes by repeated salt washes, and gp96 is not an integral membrane protein. Our observations suggest that gp96 and perhaps other HSPs are anchored to the cell surface as part of larger molecular complexes, which also transport them to the cell surface.

FAK-dependent regulation of myofibroblast differentiation

Fibroblasts and myofibroblasts both participate in wound healing. Transforming growth factor beta (TGFβ) induces fibroblasts to differentiate into myofibroblasts, whereas fibroblast growth factor and heparin (FGF/h) induce myofibroblasts to “de‐differentiate” into fibroblasts. TGFβ induces expression of smooth muscle alpha actin (SMαA) and incorporation into in stress fibers, a phenotype of differentiated myofibroblasts. Additionally, TGFβ induces the expression of fibronectin and fibronectin integrins. Fibronectin‐generated signals contribute to the TGFβ‐mediated myofibroblast differentiation. Because fibronectin signals are transmitted through focal adhesion kinase (FAK), it was predicted that FAK would be essential to TGFβ‐mediated myofibroblast differentiation. To determine whether the FAK signaling pathway is required for myofibroblast differentiation, we used two approaches to decrease FAK in mouse embryo fibroblasts (MEFs): 1) FAK +/+ MEFs, in which FAK protein expression was greatly decreased by short hairpin RNA (shRNA), and 2) FAK −/− MEFs, which lack FAK. In both cases, the majority of cells were myofibroblasts, expressing SMαA in stress fibers even after treatment with FGF/h. Furthermore, both the surface expression of FGFRs and FGF signaling were greatly reduced in FAK−/− MEFs. We conclude that FAK does not contribute to TGFβ‐dependent myofibroblast differentiation. Instead, FAK was necessary for FGF/h signaling in down‐regulating expression of SMαA, which is synonymous with myofibroblast differentiation. FAK activation could contribute to regulating myofibroblast differentiation, thereby ameliorating fibrosis.—Greenberg, R. S., Bernstein, A. M., Benezra, M., Gelman, I. H., Taliana, L., Masur, S. K. FAK‐dependent regulation of myofibroblast differentiation. FASEB J. 20, E191–E200 (2006)

Mutations in PDGFRB cause autosomal-dominant infantile myofibromatosis.

Infantile myofibromatosis (IM) is a disorder of mesenchymal proliferation characterized by the development of nonmetastasizing tumors in the skin, muscle, bone, and viscera. Occurrence within families across multiple generations is suggestive of an autosomal-dominant (AD) inheritance pattern, but autosomal-recessive (AR) modes of inheritance have also been proposed. We performed whole-exome sequencing (WES) in members of nine unrelated families clinically diagnosed with AD IM to identify the genetic origin of the disorder. In eight of the families, we identified one of two disease-causing mutations, c.1978C>A (p.Pro660Thr) and c.1681C>T (p.Arg561Cys), in PDGFRB. Intriguingly, one family did not have either of these PDGFRB mutations but all affected individuals had a c.4556T>C (p.Leu1519Pro) mutation in NOTCH3. Our studies suggest that mutations in PDGFRB are a cause of IM and highlight NOTCH3 as a candidate gene. Further studies of the crosstalk between PDGFRB and NOTCH pathways may offer new opportunities to identify mutations in other genes that result in IM and is a necessary first step toward understanding the mechanisms of both tumor growth and regression and its targeted treatment.

Reversible inhibition of endocytosis in cultured neurons from the Drosophila temperature-sensitive mutant shibirets1.

The Drosophila mutant, shibirets1 (shits1), is paralyzed at restrictive temperatures (greater than 29 degrees C) by a reversible block in synaptic transmission. Heat pulses deplete synaptic vesicles in nerve terminals and inhibit endocytic internalization of plasma membrane in garland cells and oocytes. In dissociated cultures of larval central nervous system (CNS), a temperature-sensitive defect is also expressed in shits1 neurons: at 30 degrees C, growth cone formation is retarded and neurite outgrowth is arrested. We now report that we have examined constitutive endocytosis in Drosophila CNS culture and have demonstrated directly an endocytic defect in shits1 neurons. At the permissive temperature, 20-22 degrees C, both shits1 and wild-type neurons actively endocytosed fluorescein-labelled dextran (40 KD, 5%) or horseradish peroxidase (HRP, 1%). Within 5 min, HRP was seen in vesicles, cup-shaped bodies, tubules and multivesicular bodies in neurites and cell bodies. In contrast, endocytosis was inhibited in cultures derived from the temperature-sensitive paralytic shits1 by a 15 min heat pulse (30 degrees C). Even after 30 min of HRP exposure at 30 degrees C, HRP-containing membranes were absent from almost all shits1 neurites; a minority of cell bodies had a few HRP-containing vesicles. The temperature-dependent block in endocytosis was readily reversed at 20 degrees C. Interestingly, the block was overcome by high concentration of external cations: shits1 neurons in culture actively took up HRP in numerous vesicles at 30 degrees C if 18 mM Ca2+ or Mg2+ was added to the medium. Our results support the notion that membrane recycling plays a critical role in regulating neurite outgrowth. This study also provides baseline information for further mutational analysis of the mechanism underlying the membrane cycling process in cultured neurons.

Connective tissue growth factor (CTGF) expression in the brain is a downstream effector of insulin resistance-associated promotion of Alzheimer's disease β-amyloid neuropathology

The goal of this study was to further explore potential mechanisms through which diabetogenic dietary conditions that result in promotion of insulin resistance (IR), a feature of non‐insulin dependant diabetes mellitus (type‐2 diabetes), may influence Alzheimers disease (AD). Using genome‐wide array technology, we found that connective tissue growth factor (CTGF), a gene product described previously for its involvement in diabetic fibrosis, is elevated in brain tissue in an established mouse model of diet‐induced IR. With this evidence we continued to explore the regulation of CTGF in postmortem AD brain tissue and found that CTGF expression correlated with the progression of AD clinical dementia and amyloid neuritic plaque (NP) neuropathology, but not neurofibrillary tangle (NFT) deposition. Consistent with this evidence, we also found that exposure of Tg2576 mice (a model AD‐type amyloid neuropathology) to a diabetogenic diet that promotes IR results in a ∼2‐fold elevation in CTGF steady‐state levels in the brain, coincident with a commensurate promotion of AD‐type amyloid plaque burden. Finally, using in vitro cellular models of amyloid precursor protein (APP)‐processing and Aβ generation/clearance, we confirmed that human recombinant (hr)CTGF may increase Aβ1–40 and Aβ1–42 peptide steady‐state levels, possibly through a mechanism that involves γ‐secretase activation and decreased insulin‐degrading enzyme (IDE) steady‐state levels in a MAP kinase (MAPK)/ phosphatidylinositol 3‐kinase (PI‐3K)/protein kinase‐B (AKT)1‐dependent manner. The findings in this study tentatively suggest that increased CTGF expression in the brain might be a novel biological predicative factor of AD clinical progression and neuropathology in response to dietary regimens promoting IR conditions.

Mast cell degranulating peptide binds to RBL-2H3 mast cell receptors and inhibits IgE binding

Fluorescent and biotinylated analogs of mast cell degranulating (MCD) peptide were synthesized and the labels fluoresceinisothiocyanate and N-hydroxysuccinimidobiotin were conjugated at position 1 in the MCD peptide sequence. The analogs with these moieties retained histamine-releasing activity as high as that of the parent MCD peptide in rat peritoneal mast cell assays. These labeled analogs were used in rat basophilic leukemia cells (RBL-2H3) to demonstrate by confocal microscopy and flow cytometry the specific binding of MCD peptide to mast cell receptors. Consequently MCD peptide was found to compete with and inhibit the binding of fluorescent IgE on RBL cells as monitored by flow cytometry. Thus MCD peptide may prove to be useful in the study of IgE receptor-bearing cells.

TGF-β–Stimulated CTGF Production Enhanced by Collagen and Associated with Biogenesis of a Novel 31-kDa CTGF Form in Human Corneal Fibroblasts

PURPOSE Connective tissue growth factor (CTGF) is induced by transforming growth factor-beta (TGF-β) after corneal wounding. This study addressed the role of the extracellular matrix in the induction of CTGF by TGF-β. METHODS Human corneal fibroblasts (HCFs) were grown on fibronectin (FN), vitronectin (VN), or collagen (CL) in supplemented serum-free media alone or with TGF-β1 or fibroblast growth factor plus heparin. CTGF mRNA was analyzed by qPCR and protein expression by Western blot analysis of Triton X-100 (TX-100)-soluble and TX-100-insoluble cell lysates using antibodies to N-terminal, mid, and C-terminal CTGF regions. Immunocytochemistry was performed on nonconfluent or scrape-wounded confluent HCFs. RESULTS TGF-β-treated HCFs grown on CL produced five times more 38-kDa CTGF than untreated controls (72 hours). TGF-β-treated HCFs on CL secreted twofold more CTGF than those on FN or VN. Furthermore, a 31-kDa CTGF form, lacking the N-terminal domain, was detected in Triton X-100 insoluble fractions in Western blot analysis. Immunodetectable extracellular CTGF formed linear arrays parallel to, but not colocalized with, CL or FN. It also did not colocalize with FAK, vinculin, or integrins α(v)β(3) and α(5)β(1). Intracellular CTGF was detected in the Golgi apparatus and vesicles, including endosomes. CONCLUSIONS Enhanced CTGF secretion induced by TGF-β in CL-grown cells may contribute to positive feedback in which CL is overexpressed in CTGF-induced fibrosis. N-terminal CTGF fragments in the plasma of patients with severe fibrotic disease may be a product of CTGF proteolysis that also produces the newly identified 31-kDa CTGF that remains cell associated and may have its impact by non-integrin signaling pathways.

Isolation and characterization of granules of the toad bladder

SummaryThe electron-dense granules that lie just below the apical plasma membrane of granular epithelial cells of toad urinary bladder contribute glycoproteins to that apical membrane. Also, exocytosis of granules (and tubules) elicited by antidiuretic hormone potentially doubles that apical surface, during the same period the transport changes characteristic of the hormonal response occur.Granules separated from other membrane systems of the cells provide the material to assess the importance of the granules as glycocalyx precursors and in hormone action. We used isosmotic media to effect preliminary separations by differential centrifugation. Then granules were isolated by centrifugation on self-forming gradients of Percoll of decreasing hypertonicity.We find qualitative and quantitative changes in protein composition and enzymic activities in the isolated fractions. The primary criterion for granule purification was electron microscopic morphology. In addition, polypeptide species found in the granule fraction are limited in number and quantity. The granules are enzymically and morphologically not lysosomal in nature. Granules may provide the glycoproteins of the apical glycocalyx but they differ from the isolated plasma membrane fraction enzymically, in protein composition and in proportion of esterified cholesterol.We conclude that the granules are not “average” plasma membrane precursors. Their role in the membrane properties of the toad urinary bladder may now be evaluated by characterizing permeability and other properties of the isolated organelles.

Identification of the Plasma Membrane Proteolipid Protein as a Constituent of Brain Coated Vesicles and Synaptic Plasma Membrane

Abstract: We have analyzed brain coated vesicles and synaptic plasma membrane for the presence of the plasma membrane proteolipid protein. Coated vesicles were isolated from calf brain gray matter with a final purification on Sephacryl S‐1000 and reisolated twice by chromatography to ensure homogeneity. Fractions were analyzed by gel electrophoresis, immunoblotting for clathrin heavy chain, and by electron microscopy. Using an immunoblotting assay we were able to demonstrate the presence of the plasma membrane proteolipid protein in these coated vesicles at a significant level (i.e., approximately 1% of the bilayer protein of these vesicles). Reisolation of coated vesicles did not diminish the concentration of the protein in this fraction. Removal of the clathrin coat proteins or exposure of the coated vesicles to 0.1 M Na2CO3 showed that the plasma membrane proteolipid protein is not removed during uncoating and lysis but is intrinsic to the membrane bilayer of these vesicles. These studies demonstrate that plasma membrane proteolipid protein represents a significant amount of the bilayer protein of coated vesicles, suggesting that these vesicles may be a transport vehicle for the intracellular movement of the plasma membrane proteolipid protein. Isolation of synaptic plasma membranes from adult rat brain and estimation of the plasma membrane proteolipid protein content using the immunoblotting method confirmed earlier studies that show this protein is present in this membrane fraction at high levels as well (approximately 1‐2%). The level of this protein in the synaptic plasma membrane suggests that the synaptic plasma membrane is one major site to which these vesicles may be targeted or from which the protein is being retrieved.

Notes on the Heterogeneity, Circulation, and Modification of Membranes, with Emphasis on Secretory Cells, Photoreceptors, and the Toad Bladder

In considering the origin, intracellular circulation, and turnover of cellular membranes, microscopists, for obvious reasons, focus on the movement of membrane in bulk (Fig. 1) (cf. Bennett, 1969; Novikoff and Holtzman, 1976). They think largely in terms of such established or proposed mechanisms as exocytosis and endocytosis, the formation of secretory granules from sacs and vacuoles of the Golgi sacs, and the degradation of membrane within lysosomes (see legend to Fig. 1 for details and references). From morphologic evidence, and related cytochemical and autoradiographic findings, one can make a fairly persuasive case that membranes, such as those surrounding secretory structures, or even plasma membranes: (1) originate as membranes by growth or budding from the endoplasmic reticulum (ER); (2) circulate in the cell as morphologically intact entities (although, in microscopically or cytochemically detectable aspects, such as thickness, they may be modified by the Golgi apparatus or other organelles) (3) are degraded to small molecules by bulk processes such as incorporation in autophagic vacuoles or multivesicular bodies, perhaps after complex cycling among cellular compartments (see Fig. 1 and the recent studies on the involvement of the Golgi apparatus in membrane recycling by Herzog and Farquhar, 1977, and Farquhar, 1978). Such schemes account reasonably for many observations, especially when one adds to them the likelihood that membranes of certain cellular structures—notably the inner compartments of mitochondria and plastids—may be assembled locally, rather than imported from the ER.