CCN 家族屬於胞外間質蛋白，由CCN1~6 組成。過去的研究指出，CCN1 藉由和不同的Integrin 結合來影響細胞的增殖(Proliferation)、黏附(Adhesion)、移動(Migration)、分化(Differentiation)、凋亡(Apoptosis)和生存(Survival)。當心臟受到外在傷害或在壓力過大的環境下會誘導CCN1 產生，另外，Fas/Fas Ligand (FasL)的訊號傳遞機制在心臟受損時的反應佔有重要的影響力。在本篇研究中証實CCN1 會幫助心肌細胞進行黏附，並使細胞走向凋亡，而心肌細胞凋亡的程度會隨著CCN1 濃度的增加而上升。CCN1 和FasL 個別都會使心肌細胞走向凋亡，但當兩者同時對心肌細胞作用時，出現協同作用(Synergistic Effect)。在正常的情況下，心肌細胞對FasL 反應的能力，在FasL 10 ng/ml 時已達到最大限度，但當CCN1 (2 ug/ml)同時存在的情況下，心肌細胞走向凋亡的程度隨著FasL 濃度(10 ng/ml~200 ng/ml)增加而持續上升。本篇研究進一步對CCN1 和FasL 兩者造成心肌細胞凋亡及協同作用的機轉進行探討，結果指出給予CCN1 後，心肌細胞內的Caspase 3, 9 及p38 MAPK 分子被活化的量增加; 當給予Caspase 3, 8, 9 及p38 MAPK 的抑制劑後，CCN1 及FasL 所影響的凋亡作用都會被抑制。綜合本篇結果得知，CCN1 有影響心肌細胞凋亡的能力，除此之外，還能增加心肌細胞對高濃度FasL 產生反應，並且透過Casepase 3, 8, 9 及p38 MAPK 的參予，使心肌細胞走向凋亡。

The CCN family consists of six secreted extracellular matrix (ECM) proteins,CCN1 ~ 6. Among them, CCN1 regulates cell proliferation, adhesion, migration,differentiation, apoptosis, survival and extracellular matrix production through binding with integrin receptors. In the heart, CCN1 is induced during ischemia and pressure overload. Fas/Fas ligand (FasL) signaling is often associated with cardiac injury. In this study, we prove that CCN1 can regulate adhesion in cardiomyocytes. We have demonstrated that CCN1 treatment induced apoptosis in rat cardiac muscle cells (H9c2s) in a does-dependent manner. Individually, either CCN1 or FasL induced low level of cell apoptosis. In combination, CCN1 was found to synergize with FasL in inducing H9c2 cell apoptosis. Apoptosis induced by FasL reached plateau at 10 ng/ml. Interestingly, CCN1(2 ug/ml) treatment sensitized the H9c2 cells to respond to increasing dosages of FasL beyond 10 ng/ml and up to 200 ng/ml (the highest dose tested) and resulted in double amount of cell death. In an attempt to study the molecular mechanism underlying the synergism between CCN1 and FasL, we found that caspase 3, caspase 8, caspase 9 and p38 MAPK were activated by CCN1 and were required for CCN1-induced apoptosis. In addition to signaling molecules involved in CCN1 actions, we are currently investigating the cause of the increase of sensitivity in H9c2 in response to FasL under CCN1 treatment. Our results suggested that cardiomyocytes are resistant to high doses of Fas/FasL-induced cell death, and only co-existence of CCN1 would sensitize the cardiomyocytes to effectively respond to FasL resulting in increasing cell death. Targeting CCN1 activities in a stressed heart may alleviate the damage caused by cardiac cell death.

Acehan, D., X. Jiang, et al. (2002). "Three-dimensional structure of the apoptosome:
implications for assembly, procaspase-9 binding, and activation." Mol Cell
9(2): 423-32.
Aggarwal, B. B. (2003). "Signalling pathways of the TNF superfamily: a
double-edged sword." Nat Rev Immunol 3(9): 745-56.
romotes endothelial cell survival, and induces angiogenesis in
vivo." Mol Cell Biol
Babic, A. M., C. C. Chen, et al. (1999). "Fisp12/mouse connective tissue growth
factor mediates endothelial cell adhesion and migration through integrin
alphavbeta3, p
19(4): 2958-66.
Baehrecke, E. H. (2002). "How death shapes life during development." Nat Rev Mol
Cell Biol 3(10): 779-87.
Bar, A.
, S. E. Dorfman, et al. "The pro-angiogenic factor CCN1 enhances the
re-endothelialization of biological vascularized matrices in vitro." Cardiovasc
Res 85(4): 806-13.
Battegay, E. J., E. W. Raines, et al. (1995). "TNF-alpha stimulation of fibroblast
proliferation. Dependence on platelet-derived growth factor (PDGF) secretion
and alteration of PDGF receptor expression." J Immunol 154(11): 6040-7.
Beltram
ial infarction." N Engl J Med
i, A. P., K. Urbanek, et al. (2001). "Evidence that human cardiac myocytes
divide after myocard 344(23): 1750-7.
34
Ben-Levy, R., S. Hooper, et al. (1998). "Nuclear export of the stress-activated pro
kinase p38 mediated by its substrate MAPKAP kinase-2."
tein
Curr Biol 8(19):
1049-57.
Bidere, N., H. C. Su, et al. (2006). "Genetic disorders of programmed cell death in t
immune system."
he
Annu Rev Immunol 24: 321-52.
Black, R. A., C. T. Rauch, et al. (1997). "A metalloproteinase disintegrin that releases
tumour-necrosis factor-alpha from cells." Nature 385(6618): 729-33.
Bork, P. (1993). "The modular architecture of a new family of growth regulators
related to connective tissue growth factor." FEBS Lett 327(2): 125-30.
Brigstock, D. R., C. L. Steffen, et al. (1997). "Purification and characterization of
tor." J
novel heparin-binding growth factors in uterine secretory fluids. Identification
as heparin-regulated Mr 10,000 forms of connective tissue growth fac
Biol Chem 272(32): 20275-82.
Chen, C
matricellular proteins." Int J Biochem Cell Biol
. C. and L. F. Lau (2009). "Functions and mechanisms of action of CCN
41(4): 771-83.
Chen, C. C., J. L. Young, et al. (2007). "Cytotoxicity of TNFalpha is regulated by
integrin-mediated matrix signaling." EMBO J 26(5): 1257-67.
Chen, N., C. C. Chen, et al. (2000). "Adhesion of human skin fibroblasts to Cyr61 is
mediated through integrin alpha 6beta 1 and cell surface heparan sulfate
proteoglycans." J Biol Chem 275(32): 24953-61.
35
Cheng, H. L. and E. L. Feldman (1998). "Bidirectional regulation of p38 kinase an
c-Jun N-terminal protein kinase by insulin-like growth factor-I.
d
" J Biol Chem
273(23): 14560-5.
Date, T le
perfusion injury." J Mol Cell
., S. Mochizuki, et al. (2003). "Differential effects of membrane and solub
Fas ligand on cardiomyocytes: role in ischemia/re
Cardiol 35(7): 811-21.
Degterev, A., M. Boyce, et al. (2003). "A decade of caspases." Oncogene 22(53):
8543-67.
Desagher, S. and J. C. Martinou (2000). "Mitochondria as the central control point o
apoptosis."
f
Trends Cell Biol 10(9): 369-77.
Deveraux, Q. L. and J. C. Reed (1999). "IAP family proteins--suppressors of
apoptosis." Genes Dev 13(3): 239-52.
Elmore, S. (2007). "Apoptosis: a review of programmed cell death." Toxicol Pathol
Enari, M., H. Hug, et al. (1995). "Involvement of an ICE-like protease in
35(4): 495-516.
Fas-mediated apoptosis." Nature 375(6526): 78-81.
Enari, M., R. V. Talanian, et al. (1996). "Sequential activation of ICE-like and
CPP32-like proteases during Fas-mediated apoptosis." Nature 380(6576):
723-6.
Enslen, H., J. Raingeaud, et al. (1998). "Selective activation of p38 mitogen-activated
36
protein (MAP) kinase isoforms by the MAP kinase kinases MKK3 and
MKK6." J Biol Chem 273(3): 1741-8.
Fakler, th
ood acute leukemia cells and overcome
Bcl-2-mediated resistance." Blood
M., S. Loeder, et al. (2009). "Small molecule XIAP inhibitors cooperate wi
TRAIL to induce apoptosis in childh
113(8): 1710-22.
Fantz, D t
phosphorylate specific residues." J Biol
. A., D. Jacobs, et al. (2001). "Docking sites on substrate proteins direc
extracellular signal-regulated kinase to
Chem 276(29): 27256-65.
Foltz, I
otein kinase pathway." J
. N., J. C. Lee, et al. (1997). "Hemopoietic growth factors with the exception
of interleukin-4 activate the p38 mitogen-activated pr
Biol Chem 272(6): 3296-301.
Franzen
and TRAIL-induced apoptosis." Mol Cancer Res
, C. A., C. C. Chen, et al. (2009). "Matrix protein CCN1 is critical for prostate
carcinoma cell proliferation
7(7): 1045-55.
Fuentes
nd inhibition." Biochem J
-Prior, P. and G. S. Salvesen (2004). "The protein structures that shape caspase
activity, specificity, activation a 384(Pt 2): 201-32.
Goldstein, J. C., N. J. Waterhouse, et al. (2000). "The coordinate release of
cytochrome c during apoptosis is rapid, complete and kinetically invariant."
Nat Cell Biol 2(3): 156-62.
Hanahan, D. and J. Folkman (1996). "Patterns and emerging mechanisms of the
angiogenic switch during tumorigenesis." Cell 86(3): 353-64.
37
Haunstetter, A. and S. Izumo (1998). "Apoptosis: basic mechanisms and imp
for cardiovascular disease."
lications
Circ Res 82(11): 1111-29.
Hilfiker-Kleiner, D., K. Kaminski, et al. (2004). "Regulation of proangiogenic factor
CCN1 in cardiac muscle: impact of ischemia, pressure overload, and
neurohumoral activation." Circulation 109(18): 2227-33.
Holt, G. D., M. K. Pangburn, et al. (1990). "Properdin binds to sulfatide
[Gal(3-SO4)beta 1-1 Cer] and has a sequence homology with other proteins
that bind sulfated glycoconjugates." J Biol Chem 265(5): 2852-5.
Hu, Y.,
optosis." EMBO J
M. A. Benedict, et al. (1999). "Role of cytochrome c and dATP/ATP
hydrolysis in Apaf-1-mediated caspase-9 activation and ap
18(13): 3586-95.
Igney, F. H. and P. H. Krammer (2002). "Death and anti-death: tumour resistance to
apoptosis." Nat Rev Cancer 2(4): 277-88.
Ilic, D., E. A. Almeida, et al. (1998). "Extracellular matrix survival signals tra
by focal adhesion kinase suppress p53-mediated apoptosis."
nsduced
J Cell Biol 143(2
547-60.
):
Cancer
Iyoda, K., Y. Sasaki, et al. (2003). "Involvement of the p38 mitogen-activated protein
kinase cascade in hepatocellular carcinoma." 97(12): 3017-26.
through integrin alpha(IIb)beta(3)." J Biol Chem
Jedsadayanmata, A., C. C. Chen, et al. (1999). "Activation-dependent adhesion of
human platelets to Cyr61 and Fisp12/mouse connective tissue growth factor is
mediated 274(34): 24321-7.
38
Juo, P., C. J. Kuo, et al. (1997). "Fas activation of the p38 mitogen-activated protein
kinase signalling pathway requires ICE/CED-3 family proteases." Mol Cell
Biol 17(1): 24-35.
Juric, V., C. C. Chen, et al. (2009). "Fas-mediated apoptosis is regulated by the
extracellular matrix protein CCN1 (CYR61) in vitro and in vivo." Mol Cell
Biol 29(12): 3266-79.
Kang, P
iovascular diseases." Trends in Molecular Medicine
. M. and S. Izumo (2003). "Apoptosis in heart: basic mechanisms and
implications in card 9(4):
177-182.
Kerr, J.
lications in tissue kinetics." Br J Cancer
F., A. H. Wyllie, et al. (1972). "Apoptosis: a basic biological phenomenon
with wide-ranging imp 26(4): 239-57.
tegrin alphavbeta3." J Biol Chem
Kireeva, M. L., S. C. Lam, et al. (1998). "Adhesion of human umbilical vein
endothelial cells to the immediate-early gene product Cyr61 is mediated
through in 273(5): 3090-6.
ble
Mol Cell Biol
Kireeva, M. L., F. E. Mo, et al. (1996). "Cyr61, a product of a growth factor-induci
immediate-early gene, promotes cell proliferation, migration, and adhesion."
16(4): 1326-34.
Kischk
ing complex
(DISC) with the receptor." EMBO J
el, F. C., S. Hellbardt, et al. (1995). "Cytotoxicity-dependent APO-1
(Fas/CD95)-associated proteins form a death-inducing signal
14(22): 5579-88.
Kischkel, F. C., D. A. Lawrence, et al. (2001). "Death receptor recruitment of
39
endogenous caspase-10 and apoptosis initiation in the absence of caspase-8." J
Biol Chem 276(49): 46639-46.
Krishna, M. and H. Narang (2008). "The complexity of mitogen-activated protein
kinases (MAPKs) made simple." Cell Mol Life Sci 65(22): 3525-44.
Kumar, S. (2007). "Caspase function in programmed cell death." Cell Death Differ
Kurosaka, K., M. Takahashi, et al. (2003). "Silent cleanup of very early apoptotic cells
14(1): 32-43.
by macrophages." J Immunol 171(9): 4672-9.
Kyriakis, J. M. and J. Avruch (2001). "Mammalian mitogen-activated protein kinase
signal transduction pathways activated by stress and inflammation." Physiol
Rev 81(2): 807-69.
Lau, L. F. and S. C. Lam (1999). "The CCN family of angiogenic regulators: the
integrin connection." Exp Cell Res 248(1): 44-57.
te
ptor-mediated cellular functions." J Biol Chem
Leu, S. J., N. Chen, et al. (2004). "Targeted mutagenesis of the angiogenic protein
CCN1 (CYR61). Selective inactivation of integrin alpha6beta1-heparan sulfa
proteoglycan corece 279(42):
44177-87.
Li, M. ne receptor is required for
clearance of apoptotic cells." Science
O., M. R. Sarkisian, et al. (2003). "Phosphatidylseri
302(5650): 1560-3.
Li, P., D. Nijhawan, et al. (1997). "Cytochrome c and dATP-dependent formation of
40
Apaf-1/caspase-9 complex initiates an apoptotic protease cascade." Cell 91(4
479-89.
):
rc Res
Li, Y., G. Takemura, et al. (2004). "Critical roles for the Fas/Fas ligand system in
postinfarction ventricular remodeling and heart failure." Ci 95(6):
627-36.
Liu, W.
inactivation responsible for upregulation of Fas and FasL and
autocrine Fas-mediated cell death in Taiwan cobra phospholipase A(2)-treated
H., Y. C. Cheng, et al. (2009). "ROS-mediated p38alpha MAPK activation
and ERK
U937 cells." J Cell Physiol 219(3): 642-51.
Liu, X., C. N. Kim, et al. (1996). "Induction of apoptotic program in cell-free extracts:
requirement for dATP and cytochrome c." Cell 86(1): 147-57.
Martin, D. A., R. M. Siegel, et al. (1998). "Membrane oligomerization and cleavage
activates the caspase-8 (FLICE/MACHalpha1) death signal." J Biol Chem
273(8): 4345-9.
Miranti, C. K. and J. S. Brugge (2002). "Sensing the environment: a historical
perspective on integrin signal transduction." Nat Cell Biol 4(4): E83-90.
Mo, F. E. and L. F. Lau (2006). "The matricellular protein CCN1 is essential for
cardiac development." Circ Res 99(9): 961-9.
Moss, M. L., S. L. Jin, et al. (1997). "Cloning of a disintegrin metalloproteinase that
processes precursor tumour-necrosis factor-alpha." Nature 385(6618): 733-6.
41
Muzio, M., A. M. Chinnaiyan, et al. (1996). "FLICE, a novel FADD-homologous
ICE/CED-3-like protease, is recruited to the CD
95 (Fas/APO-1)
death--inducing signaling complex." Cell 85(6): 817-27.
Muzio,
activation." J Biol Chem
M., B. R. Stockwell, et al. (1998). "An induced proximity model for caspase-8
273(5): 2926-30.
Nagata, S. (1997). "Apoptosis by death factor." Cell 88(3): 355-65.
Nagata, S. (1999). "Fas ligand-induced apoptosis." Annual Review of Genetics 33:
Nakanishi, T., Y. Kimura, et al. (1997). "Cloning of a mRNA preferentially expressed
hondrocytic cell
line that is identical with connective tissue growth factor (CTGF) mRNA."
29-55.
in chondrocytes by differential display-PCR from a human c
Biochem Biophys Res Commun 234(1): 206-10.
Narula, J., N. Haider, et al. (1996). "Apoptosis in myocytes in end-stage heart failure."
N Engl J Med 335(16): 1182-9.
, T. P., G. P. Yang, et al. (1990). "Expression of cyr61, a growth
factor-inducible immediate-early gene."
O'Brien
Mol Cell Biol 10(7): 3569-77.
Olivetti, G., R. Abbi, et al. (1997). "Apoptosis in the failing human heart." N Engl J
Med 336(16): 1131-41.
Peter, M. E., R. C. Budd, et al. (2007). "The CD95 receptor: apoptosis revisited." Cell
129(3): 447-50.
42
Porter, A. G. and R. U. Janicke (1999). "Emerging roles of caspase-3 in apoptosis."
Cell Death Differ 6(2): 99-104.
s in a TNF-alpha-dependent manner." Cell Death Differ
Probst, B. L., L. Liu, et al. "Smac mimetics increase cancer cell response to
chemotherapeutic .
s
rdiomyopathy." Cardiovasc Toxicol
Purevjav, E., D. P. Nelson, et al. (2007). "Myocardial Fas ligand expression increase
susceptibility to AZT-induced ca 7(4):
255-63.
Rainge
environmental stress cause p38 mitogen-activated protein kinase activation by
aud, J., S. Gupta, et al. (1995). "Pro-inflammatory cytokines and
dual phosphorylation on tyrosine and threonine." J Biol Chem 270(13):
7420-6.
Raman, M., W. Chen, et al. (2007). "Differential regulation and properties of
MAPKs." Oncogene 26(22): 3100-3112.
ll, J. C. and C. B. Thompson (1999). "The central effectors of cell death
immune
Rathme in the
system." Annu Rev Immunol 17: 781-828.
Rodriguez, J. and Y. Lazebnik (1999). "Caspase-9 and APAF-1 form an active
holoenzyme." Genes Dev 13(24): 3179-84.
Salvesen, G. S. and C. S. Duckett (2002). "IAP proteins: blocking the road to death's
door." Nat Rev Mol Cell Biol 3(6): 401-10.
Savill, J. and V. Fadok (2000). "Corpse clearance defines the meaning of cell death."
43
Nature 407(6805): 784-8.
Scaffidi, C., S. Fulda, et al. (1998). "Two CD95 (APO-1/Fas) signaling pathways."
EMBO J 17(6): 1675-87.
Schmitz, I., S. Kirchhoff, et al. (2000). "Regulation of death receptor-mediated
apoptosis pathways." Int J Biochem Cell Biol 32(11-12): 1123-36.
ripheral blood monocytes for Cyr61 (CCN1) and
connective tissue growth factor (CCN2): immediate-early gene products
Schober, J. M., N. Chen, et al. (2002). "Identification of integrin alpha(M)beta(2) as
an adhesion receptor on pe
expressed in atherosclerotic lesions." Blood 99(12): 4457-65.
Segarini, P. R., J. E. Nesbitt, et al. (2001). "The low density lipoprotein
receptor-related protein/alpha2-macroglobulin receptor is a receptor for
connective tissue growth factor." J Biol Chem 276(44): 40659-67.
Sprick, M. R., M. A. Weigand, et al. (2000). "FADD/MORT1 and caspase-8 are
recruited to TRAIL receptors 1 and 2 and are essential for apoptosis mediated
by TRAIL receptor 2." Immunity 12(6): 599-609.
Srinivasula, S. M., M. Ahmad, et al. (1998). "Autoactivation of procaspase-9 by
Apaf-1-mediated oligomerization." Mol Cell 1(7): 949-57.
Stephan
sion in cardiac myocytes
requires serine 727 of the STAT-1 transcription factor but not tyrosine 701." J
ou, A., T. M. Scarabelli, et al. (2001). "Induction of apoptosis and Fas
receptor/Fas ligand expression by ischemia/reperfu
Biol Chem 276(30): 28340-7.
44
Strasser r signaling in the
immune system." Immunity
, A., P. J. Jost, et al. (2009). "The many roles of FAS recepto
30(2): 180-92.
Takenaka, K., T. Moriguchi, et al. (1998). "Activation of the protein kinase p38 in the
spindle assembly checkpoint and mitotic arrest." Science 280(5363): 599-602.
Tanaka, M., T. Suda, et al. (1995). "Expression of the functional soluble form of
human fas ligand in activated lymphocytes." EMBO J 14(6): 1129-35.
Tewari, M. and V. M. Dixit (1995). "Fas- and tumor necrosis factor-induced apoptosis
is inhibited by the poxvirus crmA gene product." J Biol Chem 270(7):
3255-60.
Todorovic, V., C. C. Chen, et al. (2005). "The matrix protein CCN1 (CYR61) ind
apoptosis in fibroblasts."
uces
J Cell Biol 171(3): 559-68.
Vandervelde, S., M. J. van Luyn, et al. (2005). "Signaling factors in stem
cell-mediated repair of infarcted myocardium." J Mol Cell Cardiol 39(2
363-76.
):
mmatory lung disease."
Chest
Vandivier, R. W., P. M. Henson, et al. (2006). "Burying the dead: the impact of failed
apoptotic cell removal (efferocytosis) on chronic infla
129(6): 1673-82.
Wollert
/Fas ligand system : relation to diastolic wall stress in
volume-overload hypertrophy in vivo and activation of the transcription factor
, K. C., J. Heineke, et al. (2000). "The cardiac Fas (APO-1/CD95)
Receptor
AP-1 in cardiac myocytes." Circulation 101(10): 1172-8.
45
Xing, J
nd p38 mitogen-activated protein kinase pathways to
stimulate CREB serine 133 phosphorylation." Mol Cell Biol
., J. M. Kornhauser, et al. (1998). "Nerve growth factor activates extracellular
signal-regulated kinase a
18(4): 1946-55.
Yang, G. P. and L. F. Lau (1991). "Cyr61, product of a growth factor-inducible
immediate early gene, is associated with the extracellular matrix and the cell
surface." Cell Growth Differ 2(7): 351-7.
Yaniv, G., M. Shilkrut, et al. (2005). "Hydrogen peroxide predisposes neonatal rat
ventricular myocytes to Fas-mediated apoptosis." Biochem Biophys Res
Commun 336(3): 740-6.