Beta2-adrenergic receptor regulates cardiac fibroblast autophagy and collagen degradation

Autophagy is a physiological degradative process key to cell survival during nutrient deprivation, cell differentiation and development. It plays a major role in the turnover of damaged macromolecules and organelles, and it has been involved in the pathogenesis of different cardiovascular diseases. Activation of the adrenergic system is commonly associated with cardiac fibrosis and remodeling, and cardiac fibroblasts are key players in these processes. Whether adrenergic stimulation modulates cardiac fibroblast autophagy remains unexplored. In the present study, we aimed at this question and evaluated the effects of b₂-adrenergic stimulation upon autophagy. Cultured adult rat cardiac fibroblasts were treated with agonists or antagonists of beta-adrenergic receptors (b-AR), and autophagy was assessed by electron microscopy, GFP-LC3 subcellular distribution, and immunowesternblot of endogenous LC3. The predominant expression of b₂-ARs was determined and characterized by radioligand binding assays using [³H]dihydroalprenolol. Both, isoproterenol and norepinephrine (non-selective b-AR agonists), as well as salbutamol (selective b₂-AR agonist) increased autophagic flux, and these effects were blocked by propanolol (b-AR antagonist), ICI-118,551 (selective b₂-AR antagonist), 3-methyladenine but not by atenolol (selective b₁-AR antagonist). The increase in autophagy was correlated with an enhanced degradation of collagen, and this effect was abrogated by the inhibition of autophagic flux. Overall, our data suggest that b₂-adrenergic stimulation triggers autophagy in cardiac fibroblasts, and that this response could contribute to reduce the deleterious effects of high adrenergic stimulation upon cardiac fibrosis.     

PTEN regulates fibroblast elimination during collagen matrix contraction

During tissue repair, excess fibroblasts are eliminated by apoptosis. This physiologic process limits fibrosis and restores normal anatomic patterns. Replicating physiologic apoptosis associated with tissue repair, fibroblasts incorporated into type I collagen matrices undergo apoptosis in response to collagen matrix contraction. In this in vitro model of wound repair, fibroblasts first attach to collagen via alpha2beta1 integrin. This provides a survival signal via activation of the phosphatidylinositol 3-kinase/Akt signal pathway. However, during subsequent collagen matrix contraction, the level of phosphorylated Akt progressively declines, triggering apoptosis. The mechanism underlying the fall in phosphorylated Akt is incompletely understood. Here we show that PTEN phosphatase becomes activated during collagen matrix contraction and is responsible for antagonizing phosphatidylinositol 3-kinase activity and promoting a decline in phosphorylated Akt and fibroblast apoptosis in response to collagen contraction. PTEN null fibroblasts displayed enhanced levels of phosphorylated Akt and were resistant to collagen matrix contraction-induced apoptosis. Reconstitution of PTEN in PTEN null cells conferred susceptibility to apoptosis in response to contraction of collagen matrices. Consistent with this, knockdown of PTEN in PTEN(+/+) embryonic fibroblasts by small interfering RNA augmented Akt activity and suppressed apoptosis in contractile collagen matrices. Furthermore, inhibition of Akt activity restored the sensitivity of PTEN null cells to collagen contraction-induced apoptosis, indicating that the mechanism by which PTEN alters fibroblast viability is through modulation of phosphorylated Akt levels. Our work suggests that collagen matrix contraction activates PTEN by a mechanism involving cytoskeletal disassembly. Our studies indicate a key role for PTEN in regulating fibroblast viability during tissue repair.     

SPARC regulates processing of procollagen I and collagen fibrillogenesis in dermal fibroblasts

A characterization of the factors that control collagen fibril formation is critical for an understanding of tissue organization and the mechanisms that lead to fibrosis. SPARC (secreted protein acidic and rich in cysteine) is a counter-adhesive protein that binds collagens. Herein we show that collagen fibrils in SPARC-null skin from mice 1 month of age were inefficient in fibril aggregation and accumulated in the diameter range of 60-70 nm, a proposed intermediate in collagen fibril growth. In vitro, procollagen I produced by SPARC-null dermal fibroblasts demonstrated an initial preferential association with cell layers, in comparison to that produced by wild-type fibroblasts. However, the collagen I produced by SPARC-null cells was not efficiently incorporated into detergent-insoluble fractions. Coincident with an initial increase in cell association, greater amounts of total collagen I were present as processed forms in SPARC-null versus wild-type cells. Addition of recombinant SPARC reversed collagen I association with cell layers and decreased the processing of procollagen I in SPARC-null cells. Although collagen fibers formed on the surface of SPARC-null fibroblasts earlier than those on wild-type cells, fibers on SPARC-null fibroblasts did not persist. We conclude that SPARC mediates the association of procollagen I with cells, as well as its processing and incorporation into the extracellular matrix.     

SPARC/SFN interaction, suppresses type I collagen in dermal fibroblasts

We previously suggested that keratinocyte releasable factors might modulate the wound healing process by regulating the expression of key extracellular matrix components such as collagenase (matrix metalloproteinase-1) and type I collagen in fibroblasts. The first one, we called it keratinocyte-derived anti-fibrogenic factor (KDAF), identified as stratifin (SFN) also named 14-3-3σ, revealing a strong collagenase activity. However, the second factor, which we named keratinocyte-derived collagen-inhibiting factor(s) (KD-CIF) that has shown to control the synthesis of type I collagen, was not known. Upon conducting a series of systematic protein purification methods followed by mass spectroscopy, two proteins: secreted protein acidic rich in cystein (SPARC) and SFN were identified in keratinocyte-conditioned media. Using co-immunoprecipitation and 3D modeling, we determined that SFN and SPARC form a complex thereby controlling the type I collagen synthesis and expression in fibroblasts. The levels of these proteins in fibrotic tissues (animal and human) were also evaluated and a differential expression of these proteins between normal and fibrotic tissue confirmed their potential role in development of fibrotic condition. In conclusion, this study describes for the first time an interaction between SPARC and SFN that may have implications for the regulation of matrix deposition and prevention of dermal fibrotic conditions such as hypertrophic scars and keloid.     

Tumor cell and carcinoma-associated fibroblast interaction regulates matrix metalloproteinases and their inhibitors in oral squamous cell carcinoma

Co-culture of periodontal ligament (PDL) fibroblasts and SCC-25 oral squamous carcinoma cells (OSCC), results in conversion of PDLs into carcinoma-associated fibroblasts (CAFs). Paracrin circuits between CAFs and OSCC cells were hypothesized to regulate the gene expression of matrix remodeling enzymes in their co-culture, which was performed for 7days, followed by analysis of the mRNA/protein expression and activity of metalloproteinases (MMPs), their tissue inhibitors (TIMPs) and other relevant genes. Interleukin1-β, transforming growth factor-β1, fibronectin and αvβ6 integrin have shown to be involved in the regulation of the MMP and TIMP gene expression in co-culture of CAFs and tumor cells. In addition, these cells also cooperated in activation of MMP pro-enzymes. It is particularly interesting that the fibroblast-produced inactive MMP-2 has been activated by the tumor-cell-produced membrane-type 1 matrix metalloproteinase (MT1-MMP). The crosstalk between cancer- and the surrounding fibroblast stromal-cells is essential for the fine tuning of cancer cells invasivity.     

Regulators of cardiac fibroblast cell state

Fibroblasts are the primary regulator of cardiac extracellular matrix (ECM). In response to disease stimuli cardiac fibroblasts undergo cell state transitions to a myofibroblast phenotype, which underlies the fibrotic response in the heart and other organs. Identifying regulators of fibroblast state transitions would inform which pathways could be therapeutically modulated to tactically control maladaptive extracellular matrix remodeling. Indeed, a deeper understanding of fibroblast cell state and plasticity is necessary for controlling its fate for therapeutic benefit. p38 mitogen activated protein kinase (MAPK), which is part of the noncanonical transforming growth factor β (TGFβ) pathway, is a central regulator of fibroblast to myofibroblast cell state transitions that is activated by chemical and mechanical stress signals. Fibroblast intrinsic signaling, local and global cardiac mechanics, and multicellular interactions individually and synergistically impact these state transitions and hence the ECM, which will be reviewed here in the context of cardiac fibrosis.     

Hexarelin suppresses cardiac fibroblast proliferation and collagen synthesis in rat

Abnormal growth of cardiac fibroblasts is critically involved in the pathophysiology of cardiac hypertrophy/remodeling. Hexarelin is a synthetic growth hormone secretagogue (GHS), which possesses a variety of cardiovascular protective activities mediated via the GHS receptor (GHSR), including improving cardiac dysfunction and remodeling. The cellular and molecular mechanisms underlying the effect of GHS on cardiac fibrosis are, however, not clear. In this report, cultured cardiac fibroblasts from 8-day-old rats were stimulated with ANG II or FCS to induce proliferation. The fibroblast proliferation and DNA and collagen synthesis were evaluated utilizing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, (3)H-thymidine incorporation, and (3)H-proline incorporation. The level of mRNA of transforming growth factor (TGF)-beta was evaluated by RT-PCR, and the active TGF-beta1 release from cardiac fibroblasts was evaluated by ELISA. The level of cellular cAMP was measured by radioimmunoassay. In addition, the effects of 3,7-dimethyl-l-propargylxanthine (DMPX; a specific adenosine receptor A(2)R antagonist) and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; a specific A(1)R antagonist) were tested. It was found that incubation with 10(-7) mol/l hexarelin for 24 h 1) inhibited the ANG II-induced proliferation and collagen synthesis and the 5% FCS- and TGF-beta-induced increase of DNA synthesis in cardiac fibroblast and 2) reduced ANG II-induced upregulation of TGF-beta mRNA expression and active TGF-beta1 release from fibroblasts. Hexarelin increased the cellular level of cAMP in cardiac fibroblasts. DMPX (10(-8) mol/l) but not DPCPX abolished the effect of hexarelin on cardiac fibroblast DNA synthesis. It is concluded that hexarelin inhibits DNA and collagen synthesis and proliferation of cardiac fibroblasts through activation of both GHSR and A(2)R and diminishment of ANG II-induced increase in TGF-beta expression and release.     

IGF-1 regulates cardiac fibroblast apoptosis induced by osmotic stress

In this study we have determined the ability of IGF-1 to protect cardiac fibroblasts against osmotic-induced apoptosis and investigated the potential mechanism(s) underlying this protection. Treatment with IGF-1 (1-100 ng/ml) promoted a dose dependent increase in cell survival against osmotic cell death. Both Akt and ERK1/2 were rapidly phosphorylated by IGF-1 and blocked by wortmannin and PD98059, inhibitors of their upstream activators respectively. However, IGF-1-induced protection was mediated via a wortmannin-dependent but PD98059-independent pathway as determined by cell survival assay suggesting a role of PI3-K/Akt. Furthermore, IGF-1 appeared to reduce the activation of a number of early components in the apoptotic pathway in a wortmannin dependent manner including the osmotic stress-induced perturbation in mitochondrial membrane potential, cleavage and activation of caspase-3 and DNA fragmentation. Thus, the results suggest that IGF-1 regulates osmotic stress-induced apoptosis via the activation of the PI3-K/Akt pathway at a point upstream of the mitochondria and caspase-3.     

SPARC interacts with AMPK and regulates GLUT4 expression

AMP-activated protein kinase (AMPK) is a critical regulator of glucose metabolism. To elucidate the biochemical mechanisms by which AMPK regulates glucose and fat metabolism, we conducted a yeast two-hybrid screen to identify its interacting partners. A yeast two-hybrid system was used to screen a mouse embryo cDNA library for proteins able to bind mouse AMPKα1. We also demonstrated an endogenous interaction between AMPKα1 and its interacting partner by co-immunoprecipitation of the endogenous proteins using specific antibodies in HepG2 cells, and in rat kidney, liver, skeletal muscle, and fat tissue. We show that secreted protein acidic and rich in cysteine (SPARC) is an AMPK-interacting protein, and the two proteins enhance each other. AMPK activation increases SPARC expression, and knockdown of AMPK to inhibit endogenous AMPK expression reduces SPARC protein levels. On the other hand, SPARC siRNA reduces AICAR-stimulated AMPK phosphorylation. SPARC affects AMPK-mediated glucose metabolism through regulation of Glut4 expression in L6 myocytes. Our findings suggest that SPARC may be involved in regulating glucose metabolism via AMPK activation. These results provide a starting point for efforts to clarify the relationship between AMPK and SPARC, and deepen our understanding of their roles in fat and glucose metabolism.     

Quantitative study of cell interaction with collagen and fibronectin

The interaction of BHK-fibroblasts with collagen or fibronectin-collagen complex was investigated quantitatively. For that purpose an improved method for production of defined cell substrata was developed. The method permitted reproducible coupling of different ligands to glass via an amino or carboxyl group. BHK-cells grown on collagen required a minimum density of 15-20 ng collagen/cm2 for spreading. When grown on fibronectin adsorbed on collagen the cells were found to remove fibronectin from the substratum at a rate of 0.15 pg/(cell X h).     

Cell-cycle dependent tyrosine phosphorylation on mortalin regulates its interaction with fibroblast growth factor-1

We previously reported that endogenously expressed, intracellularly localized fibroblast growth factor (FGF)-1 interacts with mortalin. Here we report that FGF-1 added to the culture medium of quiescent BALB/c3T3 cells is taken up by the cells and interacts with mortalin in the cells in a regulated manner. Although both the internalized FGF-1 and mortalin were present at high levels throughout the FGF-1-initiated cell cycle, their interaction became apparent only in late G1 phase. Interestingly, mortalin was preferentially tyrosine phosphorylated at the same time, and when its normally weak phosphorylation in early G1 phase was augmented by treating the cells with vanadate, a strong interaction between mortalin and FGF-1 was established. Conversely, when phosphorylated mortalin was treated with tyrosine phosphatase, its interaction with FGF-1 was abrogated. These results indicate that FGF-1 taken up by cells preferentially interacts with mortalin in late G1 phase of the cell cycle, and that tyrosine phosphorylation of mortalin regulates this interaction.     

Effects of collagen density on cardiac fibroblast behavior and gene expression

Interactions between cells and the extracellular matrix (ECM) play essential roles in modulating cell behavior during development and disease. The myocardial ECM is composed predominantly of interstitial collagen type I and type III. The composition, organization, and accumulation of these collagens are altered concurrent with cardiovascular development and disease. Changes in these parameters are thought to play significant roles in myocardial function. While a number of studies have examined how changes in the ECM affect myocardial function as a whole, much less is known regarding the response at the cellular level to changes in the collagenous ECM. Experiments were carried out to determine the effects of alterations in collagen density and ECM stiffness on the behavior of isolated heart fibroblasts. In vitro bioassays were performed to measure the effects of changes in collagen concentration (0.75-1.25 mg/ml) on adhesion, migration, spreading, and gene expression by heart fibroblasts. Increased density of collagen in 3-dimensional gels resulted in more efficient adhesion, spreading, and migration by heart fibroblasts. These experiments indicated that the density of the collagen matrix has a significant impact on fibroblast function. These studies begin to elucidate the effects of ECM density at the cellular level in the myocardium.     

Cadherin-mediated cell interaction regulates germ cell determination in mice

The germ cell lineage segregates from the somatic cell lineages in early embryos. Germ cell determination in mice is not regulated by maternally inherited germplasm, but is initiated within the embryo during gastrulation. However, the mechanisms of germ cell specification in mice remain unknown. We located precursors to primordial germ cells (PGCs) within early embryos, and show here that cell-cell interaction among these precursors is required for germ cell specification. We found that the expression of a calcium-dependent cell adhesion molecule, E-cadherin, is restricted to the proximal region of extra-embryonic mesoderm that contains PGC precursors, and that blocking the functions of E-cadherin with an antibody inhibits PGC formation in vitro. These results showed that E-cadherin-mediated cell-cell interaction among cells containing PGC precursors is essential to directing such cells to the germ cell fate.     

Lysine hydroxylation of collagen in a fibroblast cell culture system

The lysine (Lys) hydroxylation pattern of type I collagen produced by human fibroblasts in culture was analyzed and compared. Fibroblasts were cultured from normal human skin (NSF), keloid (KDF), fetal skin (FDF), and skin tissues of Ehlers-Danlos syndrome type VIA and VIB patients (EDS-VIA and -VIB). The type I collagen alpha chains with or without non-helical telopeptides were purified from the insoluble matrix and analyzed. In comparison with NSFs, KDF and FDF showed significantly higher Lys hydroxylation, particularly in the telopeptide domains of both alpha chains. Both EDS-VIA and -VIB showed markedly lower Lys hydroxylation in the helical domains of both alpha chains whereas that in the telopeptides was comparable with those of NSFs. A similar profile was observed in the tissue sample of the EDS-VIB patient. These results demonstrate that the Lys hydroxylation pattern is domain-specific within the collagen molecule and that this method is useful to characterize the cell phenotypes in normal/pathological connective tissues.     

SPARC regulates cell cycle progression in mesangial cells via its inhibition of IGF-dependent signaling

Glomerular mesangial cells both synthesize and respond to insulin-like growth factor-1 (IGF-1). Increased activity of the IGF signaling pathway has been implicated as a major contributor to renal enlargement and subsequent development of diabetic nephropathy. Secreted protein acidic and rich in cysteine (SPARC), a matricellular protein, has been shown to modulate the interaction of cells with growth factors and extracellular matrix. We have reported that primary glomerular mesangial cells derived from SPARC-null mice exhibit an accelerated rate of proliferation and produce substantially decreased levels of transforming growth factor beta1 (TGF-beta1) in comparison to their wild-type counterparts (Francki et al. [1999] J. Biol. Chem. 274: 32145-32152). Herein we present evidence that SPARC modulates IGF-dependent signaling in glomerular mesangial cells. SPARC-null mesangial cells produce increased amounts of IGF-1 and -2, as well as IGF-1 receptor (IGF-1R) in comparison to wild-type cells. Addition of recombinant SPARC to SPARC-null cells inhibited IGF-1-stimulated mitogen activated protein kinase (MAPK) activation and DNA synthesis. We also show that the observed accelerated rate of basal and IGF-1-stimulated proliferation in mesangial cells derived from SPARC-null animals is due, at least in part, to markedly diminished levels of cyclin D1 and the cyclin-dependent kinase (cdk) inhibitors p21 and p27. Since expression of SPARC in the glomerulus is especially prominent during renal injury, our findings substantiate previous claims that SPARC is involved in glomerular remodeling and repair, a process commonly associated with mesangioproliferative glomerulonephritis and diabetic nephropathy.     

Hippocampal SPARC regulates depression‐related behavior

SPARC (secreted protein acidic and rich in cysteine) is a matricellular protein highly expressed during development, reorganization and tissue repair. In the central nervous system, glial cells express SPARC during development and in neurogenic regions of the adult brain. Astrocytes control the glutamate receptor levels in the developing hippocampus through SPARC secretion. To further characterize the role of SPARC in the brain, we analyzed the hippocampal‐dependent adult behavior of SPARC KO mice. We found that SPARC KO mice show increased levels of anxiety‐related behaviors and reduced levels of depression‐related behaviors. The antidepressant‐like phenotype could be rescued by adenoviral vector‐mediated expression of SPARC in the adult hippocampus, but anxiety‐related behavior persisted in these mice. To identify the cellular mechanisms underlying these behavioral alterations, we analyzed neuronal activity and neurogenesis in the dentate gyrus (DG). SPARC KO mice have increased levels of neuronal activity, evidenced as more neurons that express c‐Fos after a footshock. SPARC also affects cell proliferation in the subgranular zone of the DG, although it does not affect maturation and survival of new neurons. SPARC expression in the adult DG does not revert the proliferation phenotype in KO mice, but our results suggest a role of SPARC in limiting the survival of new neurons in the DG. This work suggests that SPARC could affect anxiety‐related behavior by modulating neuronal activity, and that depression‐related behavior is dependent upon the adult expression of SPARC, which affects adult brain function by mechanisms that need to be elucidated.     

MiR‐590‐3p regulates proliferation,migration and collagen synthesis of cardiac fibroblast by targeting ZEB1

Previous studies have implicated the attractive and promising role of miR‐590‐3p to restore the cardiac function following myocardial infarction (MI). However, the molecular mechanisms for how miR‐590‐3p involves in cardiac fibrosis remain largely unexplored. Using human cardiac fibroblasts (HCFs) as the cellular model, luciferase report assay, mutation, EdU assay and transwell migration assay were applied to investigate the biological effects of miR‐590‐3p on the proliferation, differentiation, migration and collagen synthesis of cardiac fibroblasts. We found that miR‐590‐3p significantly suppressed cell proliferation and migration of HCFs. The mRNA and protein expression levels of α‐SMA, Col1A1 and Col3A were significantly decreased by miR‐590‐3p. Moreover, miR‐590‐3p directly targeted at the 3’UTR of ZEB1 to repress the translation of ZEB1. Interfering with the expression of ZEB1 significantly decreased the cell proliferation, migration activity, mRNA and protein expressions of α‐SMA, Col1A1 and Col3A. Furthermore, the expressions of miR‐590‐3p and ZEB1 were identified in infarct area of MI model in pigs. Collectively, miR‐590‐3p suppresses the cell proliferation, differentiation, migration and collagen synthesis of cardiac fibroblasts by targeting ZEB1. These works will provide useful biological information for future studies on potential roles of miR‐590‐3p as the therapeutic target to recover cardiac function following MI.     

The interaction of Sendai virus glycoprotein-bearing recombinant vesicles with cell surfaces

Sendai virus glycoproteins HN and F were purified by immunoaffinity chromatography from virions disrupted by beta-D-octylglucoside. The purified glycoproteins were reconstituted in recombinant vesicles with phosphatidylcholine or phosphatidylethanolamine and phosphatidylserine. P815 or EL-4 cells treated with glycoprotein HN/F-phosphatidylcholine recombinant vesicles acquired the glycoproteins and retained them in the plasma membrane for 4 h as demonstrated by surface immunofluorescence specific for each protein. Cells treated with glycoprotein HN-phosphatidylcholine recombinant vesicles initially bore glycoprotein HN on the surface but the protein eluted within 2 h. Surfaces of cells treated with glycoprotein F-phosphatidylcholine recombinant vesicles did not acquire the glycoprotein. Cells treated with glycoprotein HN-phosphatidylethanolamine: phosphatidylserine recombinant vesicles or glycoprotein F-phosphatidylethanolamine: phosphatidylserine recombinant vesicles in the presence of 5 mM Ca2+ acquired each protein for at least 2 h. Experiments showed that the acquired glycoproteins capped with antibody and that when glycoproteins HN and F were together on the surface they co-capped. Acquired viral glycoproteins did not co-cap with intrinsic H-2 glycoproteins.