SPARC, a secreted protein associated with cellular proliferation, inhibits cell spreading in vitro and exhibits Ca+2-dependent binding to the extracellular matrix

SPARC (Secreted Protein Acidic and Rich in Cysteine) is a Ca+2-binding glycoprotein that is differentially associated with morphogenesis, remodeling, cellular migration, and proliferation. We show here that exogenous SPARC, added to cells in culture, was associated with profound changes in cell shape, caused rapid, partial detachment of a confluent monolayer, and inhibited spreading of newly plated cells. Bovine aortic endothelial cells, exposed to 2-40 micrograms SPARC/ml per 2 x 10(6) cells, exhibited a rounded morphology in a dose-dependent manner but remained attached to plastic or collagen-coated surfaces. These round cells synthesized protein, uniformly excluded trypan blue, and grew in aggregates after replating in media without SPARC. SPARC caused rounding of bovine endothelial cells, fibroblasts, and smooth muscle cells; however, the cell lines F9, PYS-2, and 3T3 were not affected. The activity of native SPARC was inhibited by heat denaturation and prior incubation with anti-SPARC IgG. The effect of SPARC on endothelial cells appeared to be independent of the rounding phenomenon produced by the peptide GRGDSP. Immunofluorescence localization of SPARC on endothelial cells showed preferential distribution at the leading edges of membranous extensions. SPARC bound Ca+2 in both amino- and carboxyl-terminal (EF-hand) domains and required this cation for maintenance of native structure. Solid-phase binding assays indicated a preferential affinity of native SPARC for several proteins comprising the extracellular matrix, including types III and V collagen, and thrombospondin. This binding was saturable, Ca+2 dependent, and inhibited by anti-SPARC IgG. Endothelial cells also failed to spread on a substrate of native type III collagen complexed with SPARC. We propose that SPARC is an extracellular modulator of Ca+2 and cation-sensitive proteins or proteinases, which facilitates changes in cellular shape and disengagement of cells from the extracellular matrix.     

Epithelial morphogenesis in hydra requires de novo expression of extracellular matrix components and matrix metalloproteinases

As a member of the phylum Cnidaria, the body wall of hydra is organized as an epithelium bilayer (ectoderm and endoderm) with an intervening extracellular matrix (ECM). Previous studies have established the general molecular structure of hydra ECM and indicate that it is organized as two subepithelial zones that contain basement membrane components such as laminin and a central fibrous zone that contains interstitial matrix components such as a unique type I fibrillar collagen. Because of its simple structure and high regenerative capacity, hydra has been used as a developmental model to study cell-ECM interaction during epithelial morphogenesis. The current study extends previous studies by focusing on the relationship of ECM biogenesis to epithelial morphogenesis in hydra, as monitored during head regeneration or after simple incision of the epithelium. Histological studies indicated that decapitation or incision of the body column resulted in an immediate retraction of the ECM at the wound site followed by a re-fusion of the bilayer within 1 hour. After changes in the morphology of epithelial cells at the regenerating pole, initiation of de novo biogenesis of an ECM began within hours while full reformation of the mature matrix required approximately 2 days. These processes were monitored using probes to three matrix or matrix-associated components: basement membrane-associated hydra laminin beta1 chain (HLM-beta1), interstitial matrix-associated hydra fibrillar collagen (Hcol-I) and hydra matrix metalloproteinase (HMMP). While upregulation of mRNA for both HLM-beta1 and Hcol-I occurred by 3 hours, expression of the former was restricted to the endoderm and expression of the latter was restricted to the ectoderm. Upregulation of HMMP mRNA was also associated with the endoderm and its expression paralleled that for HLM-beta1. As monitored by immunofluorescence, HLM-beta1 protein first appeared in each of the two subepithelial zones (basal lamina) at about 7 hours, while Hcol-I protein was first observed in the central fibrous zone (interstitial matrix) between 15 and 24 hours. The same temporal and spatial expression pattern for these matrix and matrix-associated components was observed during incision of the body column, thus indicating that these processes are a common feature of the epithelium in hydra. The correlation of loss of the ECM, cell shape changes and subsequent de novo biogenesis of matrix and matrix-associated components were all functionally coupled by antisense experiments in which translation of HLM-beta1 and HMMP was blocked and head regeneration was reversibly inhibited. In addition, inhibition of translation of HLM-beta1 caused an inhibition in the appearance of Hcol-I into the ECM, thus suggesting that binding of HLM-beta1 to the basal plasma membrane of ectodermal cells signaled the subsequent discharge of Hcol-I from this cell layer into the newly forming matrix. Given the early divergence of hydra, these studies point to the fundamental importance of cell-ECM interactions during epithelial morphogenesis.     

The proto-oncogene int-1 encodes a secreted protein associated with the extracellular matrix.

The proto-oncogene int-1 plays an important role in mammary tumorigenesis when activated by proviral insertions of the mouse mammary tumor virus. In normal mouse tissues the gene is expressed in the embryonic neural tube, suggesting a developmental function, while in Drosophila the homolog of int-1 is the segment polarity gene wingless. In order to study the protein products of int-1 we have derived fibroblast cell lines infected with multiple copies of a retroviral vector expressing int-1 cDNA. By Western blot analysis and immunoprecipitation we have identified a 44 kd form of int-1 protein which is secreted from these cells. The 44 kd species is distinct from the major intracellular forms of int-1 protein as judged by its slower mobility in SDS-polyacrylamide gels and by its longer half-life in pulse-chase experiments. Under normal growth conditions, little or none of the 44 kd protein is detectable in the cell culture medium but instead the majority is found associated with the extracellular matrix (ECM). The protein appears to bind heparin in vitro, suggesting that it might bind glycosaminoglycans in the ECM. These data support the view that int-1 protein may play a role in cell-cell communication over short distances.     

Ucma, a novel secreted cartilage-specific protein with implications in osteogenesis

Here we report on the structure, expression, and function of a novel cartilage-specific gene coding for a 17-kDa small, highly charged, and secreted protein that we termed Ucma (unique cartilage matrix-associated protein). The protein is processed by a furin-like protease into an N-terminal peptide of 37 amino acids and a C-terminal fragment (Ucma-C) of 74 amino acids. Ucma is highly conserved between mouse, rat, human, dog, clawed frog, and zebrafish, but has no homology to other known proteins. Remarkable are 1-2 tyrosine sulfate residues/molecule and dense clusters of acidic and basic residues in the C-terminal part. In the developing mouse skeleton Ucma mRNA is expressed in resting chondrocytes in the distal and peripheral zones of epiphyseal and vertebral cartilage. Ucma is secreted into the extracellular matrix as an uncleaved precursor and shows the same restricted distribution pattern in cartilage as Ucma mRNA. In contrast, antibodies prepared against the processed C-terminal fragment located Ucma-C in the entire cartilage matrix, indicating that it either diffuses or is retained until chondrocytes reach hypertrophy. During differentiation of an MC615 chondrocyte subclone in vitro, Ucma expression parallels largely the expression of collagen II and decreases with maturation toward hypertrophic cells. Recombinant Ucma-C does not affect expression of chondrocyte-specific genes or proliferation of chondrocytes, but interferes with osteogenic differentiation of primary osteoblasts, mesenchymal stem cells, and MC3T3-E1 pre-osteoblasts. These findings suggest that Ucma may be involved in the negative control of osteogenic differentiation of osteochondrogenic precursor cells in peripheral zones of fetal cartilage and at the cartilage-bone interface.     

Hyperoxia induces Egr-1 expression through activation of extracellular signal-regulated kinase 1/2 pathway

Early growth response gene (Egr-1) is a stress response gene activated by various forms of stress and growth factor signaling. We report that supraphysiologic concentrations of O(2) (hyperoxia) induced Egr-1 mRNA and protein expression in cultured alveolar epithelial cells, as well as in mouse lung in vivo. The contribution of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK), p38 MAPK and PI3-kinase pathways to the activation of Egr-1 in response to hyperoxia was examined. Exposure to hyperoxia resulted in a rapid phosphorylation of ERK 1/2 kinases in mouse alveolar epithelial cells LA4. MEK inhibitor PD98059, but not inhibitors of p38 MAPK or PI3-kinase pathway, prevented Egr-1 induction by hyperoxia. The signaling cascade preceding Egr-1 activation was traced to epidermal growth factor receptor (EGFR) signaling. Hyperoxia is used as supplemental therapy in some diseases and typically results in elevated levels of reactive oxygen intermediates (ROI) in many lung cell types, the organ that receives highest O(2) exposure. Our results support a pathway for the hyperoxia response that involves EGF receptor, MEK/ERK pathway, and other unknown signaling components leading to Egr-1 induction. This forms a foundation for analysis of detailed mechanisms underlying Egr-1 activation during hyperoxia and understanding its consequences for regulating cell response to oxygen toxicity.     

FIZZ1, a novel cysteine-rich secreted protein associated with pulmonary inflammation, defines a new gene family

Bronchoalveolar lavage fluid from mice with experimentally induced allergic pulmonary inflammation contains a novel 9.4 kDa cysteine-rich secreted protein, FIZZ1 (found in inflammatory zone). Murine (m) FIZZ1 is the founding member of a new gene family including two other murine genes expressed, respectively, in intestinal crypt epithelium and white adipose tissue, and two related human genes. In control mice, FIZZ1 mRNA and protein expression occur at low levels in a subset of bronchial epithelial cells and in non-neuronal cells adjacent to neurovascular bundles in the peribronchial stroma, and in the wall of the large and small bowel. During allergic pulmonary inflammation, mFIZZ1 expression markedly increases in hypertrophic, hyperplastic bronchial epithelium and appears in type II alveolar pneumocytes. In vitro, recombinant mFIZZ1 inhibits the nerve growth factor (NGF)-mediated survival of rat embryonic day 14 dorsal root ganglion (DRG) neurons and NGF-induced CGRP gene expression in adult rat DRG neurons. In vivo, FIZZ1 may modulate the function of neurons innervating the bronchial tree, thereby altering the local tissue response to allergic pulmonary inflammation.     

Imbalance between matrix metalloproteinases and tissue inhibitor of metalloproteinases in hypertensive vascular remodeling

Structural vascular changes in two-kidney, one-clip (2K-1C) hypertension may result from increased matrix metalloproteinase (MMP)-2 activity. MMP-2 activation is regulated by other MMPs, including transmembrane-MMPs, and by tissue inhibitors of MMPs (TIMPs). We have investigated the localization of MMP-2, -9, -14, and TIMPs 1–4 in hypertensive aortas and measured their levels by zymography/Western blotting and immunohistochemistry. Gelatinolytic activity was assayed in tissues by in situ zymography. Sham-operated and 2K-1C hypertensive rats were treated with doxycycline (or vehicle) for 8 weeks, and the systolic blood pressure was monitored weekly. Doxycycline attenuated 2K-1C hypertension (165 ± 11.7 mmHg versus 213 ± 7.9 mm Hg in hypertensive controls, P < 0.01), and completely prevented increase in the thicknesses of the media and the intima in 2K-1C animals (P < 0.01). Increased amounts of MMP-2, -9, and -14 were found in hypertensive aortas, as well as enhanced gelatinolytic activity. A gradient in the localization of MMP-2, -9, and -14 was found, with increased amounts detected in the intima, at sites with higher gelatinolytic activity. Doxycycline attenuated hypertension induced increases in all the 3 investigated MMPs in both the media and the intima (all P < 0.05), but it did not change the amounts of TIMPs 1–4 (P > 0.05). Therefore, an imbalance between increased amounts of MMPs at the tissue level without a corresponding increase in the quantities of TIMPs, particularly in the intima and inner media layers, appears to account for the increased proteolytic activity found in 2K-1C hypertension-induced maladaptive vascular remodeling.     

Exendin-4, a GLP-1 receptor agonist, directly induces adiponectin expression through protein kinase A pathway and prevents inflammatory adipokine expression

Exendin-4 (Ex-4) is a glucagon-like peptide-1 receptor (GLP-1R) agonist that has been used as a drug injected subcutaneously for treatment of type 2 diabetes. Many studies have revealed molecular targets of Ex-4, but its influence on adipokines has not been determined. Our study showed that Ex-4 induced secretion of adiponectin into the culture medium of 3T3-L1 adipocytes. This effect of Ex-4 is due to increased adiponectin mRNA level through the GLP-1R. Both forskolin and 3-isobutyl-1-methylxanthine (IBMX), which may finally elevate cyclic adenosine monophosphate (cAMP) concentration, prevented the induction of adiponectin expression by Ex-4. Moreover, H89, a protein kinase A inhibitor, blocked the effect of Ex-4 on adiponectin. On the other hand, Ex-4 decreased the mRNA levels of inflammatory adipokines. The results indicate that Ex-4 directly promotes adiponectin secretion via the protein kinase A pathway in 3T3-L1 adipocytes and may ameliorate insulin resistance.     

Na/H exchange regulatory factor 1, a novel AKT-associating protein, regulates extracellular signal-regulated kinase signaling through a B-Raf-mediated pathway

Na/H exchange regulatory factor 1 (NHERF1) is a scaffolding protein that regulates signaling and trafficking of several G protein-coupled receptors (GPCRs), including the parathyroid hormone receptor (PTH1R). GPCRs activate extracellular signal-regulated kinase (ERK)1/2 through different mechanisms. Here, we characterized NHERF1 regulation of PTH1R-stimulated ERK1/2. Parathyroid hormone (PTH) stimulated ERK1/2 phosphorylation by a protein kinase A (PKA)-dependent, but protein kinase C-, cyclic adenosine 5'-monophosphate-, and Rap1-independent pathway in Chinese hamster ovary cells stably transfected with the PTH1R and engineered to express NHERF1 under the control of tetracycline. NHERF1 blocked PTH-induced ERK1/2 phosphorylation downstream of PKA. This suggested that NHERF1 inhibitory effects on ERK1/2 occur at a postreceptor locus. Forskolin activated ERK1/2, and this effect was blocked by NHERF1. NHERF1 interacted with AKT and inhibited ERK1/2 activation by decreasing the stimulatory effect of 14-3-3 binding to B-Raf, while increasing the inhibitory influence of AKT negative regulation on ERK1/2 activation. This novel regulatory mechanism provides a new model by which cytoplasmic adapter proteins modulate ERK1/2 activation through a receptor-independent mechanism involving B-Raf.     

Constitutive activation of a CC-NB-LRR protein alters morphogenesis through the cytokinin pathway in Arabidopsis

Possible links between plant defense responses and morphogenesis have been postulated, but their molecular nature remains unknown. Here, we introduce the Arabidopsis semi-dominant mutant uni-1D with morphological defects. UNI encodes a coiled-coil nucleotide-binding leucine-rich-repeat protein that belongs to the disease resistance (R) protein family involved in pathogen recognition. The uni-1D mutation causes the constitutive activation of the protein, which is stabilized by the RAR1 function in a similar way as in other R proteins. The uni-1D mutation induces the upregulation of the Pathogenesis-related gene via the accumulation of salicylic acid, and evokes some of the morphological defects through the accumulation of cytokinin. The rin4 knock-down mutation, which causes the constitutive activation of two R proteins, RPS2 and RPM1, induces an upregulation of cytokinin-responsive genes and morphological defects similar to the uni-1D mutation, indicating that the constitutive activation of some R proteins alters morphogenesis through the cytokinin pathway. From these data, we propose that the modification of the cytokinin pathway might be involved in some R protein-mediated responses.     

ALK1 heterozygosity increases extracellular matrix protein expression,proliferation and migration in fibroblasts

Fibrosis is a pathological situation in which excessive amounts of extracellular matrix (ECM) are deposited in the tissue. Myofibroblasts play a crucial role in the development and progress of fibrosis as they actively synthesize ECM components such as collagen I, fibronectin and connective tissue growth factor (CTGF) and cause organ fibrosis. Transforming growth factor beta 1 (TGF-β1) plays a major role in tissue fibrosis. Activin receptor-like kinase 1 (ALK1) is a type I receptor of TGF-β1 with an important role in angiogenesis whose function in cellular biology and TGF-β signaling is well known in endothelial cells, but its role in fibroblast biology and its contribution to fibrosis is poorly studied. We have recently demonstrated that ALK1 regulates ECM protein expression in a mouse model of obstructive nephropathy. Our aim was to evaluate the role of ALK1 in several processes involved in fibrosis such as ECM protein expression, proliferation and migration in ALK1^+/+ and ALK1^+/− mouse embryonic fibroblasts (MEFs) after TGF-β1 stimulations and inhibitors. ALK1 heterozygous MEFs show increased expression of ECM proteins (collagen I, fibronectin and CTGF/CCN2), cell proliferation and migration due to an alteration of TGF-β/Smad signaling. ALK1 heterozygous disruption shows an increase of Smad2 and Smad3 phosphorylation that explains the increases in CTGF/CCN2, fibronectin and collagen I, proliferation and cell motility observed in these cells. Therefore, we suggest that ALK1 plays an important role in the regulation of ECM protein expression, proliferation and migration.     

Defective extracellular matrix remodeling in brown adipose tissue is associated with fibro-inflammation and reduced diet-induced thermogenesis

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Macrophage colony-stimulating factor induces the expression of mitogen-activated protein kinase phosphatase-1 through a protein kinase C-dependent pathway.

M-CSF triggers the activation of extracellular signal-regulated protein kinases (ERK)-1/2. We show that inhibition of this pathway leads to the arrest of bone marrow macrophages at the G0/G1 phase of the cell cycle without inducing apoptosis. M-CSF induces the transient expression of mitogen-activated protein kinase phosphatase-1 (MKP-1), which correlates with the inactivation of ERK-1/2. Because the time course of ERK activation must be finely controlled to induce cell proliferation, we studied the mechanisms involved in the induction of MKP-1 by M-CSF. Activation of ERK-1/2 is not required for this event. Therefore, M-CSF activates ERK-1/2 and induces MKP-1 expression through different pathways. The use of two protein kinase C (PKC) inhibitors (GF109203X and calphostin C) revealed that M-CSF induces MKP-1 expression through a PKC-dependent pathway. We analyzed the expression of different PKC isoforms in bone marrow macrophages, and we only detected PKCbetaI, PKCepsilon, and PKCzeta. PKCzeta is not inhibited by GF109203X/calphostin C. Of the other two isoforms, PKCepsilon is the best candidate to mediate MKP-1 induction. Prolonged exposure to PMA slightly inhibits MKP-1 expression in response to M-CSF. In bone marrow macrophages, this treatment leads to a complete depletion of PKCbetaI, but only a partial down-regulation of PKCepsilon. Moreover, no translocation of PKCbetaI or PKCzeta from the cytosol to particulate fractions was detected in response to M-CSF, whereas PKCepsilon was constitutively present at the membrane and underwent significant activation in M-CSF-stimulated macrophages. In conclusion, we remark the role of PKC, probably isoform epsilon, in the negative control of ERK-1/2 through the induction of their specific phosphatase.     

TG2, a novel extracellular protein with multiple functions

TG2 is multifunctional enzyme which can be secreted to the cell surface by an unknown mechanism where its Ca(2+)-dependent transamidase activity is implicated in a number of events important to cell behaviour. However, this activity may only be transient due to the oxidation of the enzyme in the extracellular environment including its reaction with NO probably accounting for its many other roles, which are transamidation independent. In this review, we discuss the novel roles of TG2 at the cell surface and in the ECM acting either as a transamidating enzyme or as an extracellular scaffold protein involved in cell adhesion. Such roles include its ability to act as an FN co-receptor for β integrins or in a heterocomplex with FN interacting with the cell surface heparan sulphate proteoglycan syndecan-4 leading to activation of PKCα. These different properties of TG2 involve this protein in various physiological processes, which if not regulated appropriately can also lead to its involvement in a number of diseases. These include metastatic cancer, tissue fibrosis and coeliac disease, thus increasing its attractiveness as both a therapeutic target and diagnostic marker.