About coffee, cappuccino and connective tissue growth factor-Or how to protect your liver!?

Abstract

Several epidemiological studies suggest that coffee drinking is inversely correlated with the risk of development of liver fibrosis. However, a causal, mechanistic explanation has long been pending. New results indicate that the methylxanthine caffeine, major component of coffee and the most widely consumed pharmacologically active substance in the world, might be responsible for this phenomenon as it, and even more potently its derived primary metabolite paraxanthine, inhibits transforming growth factor (TGF)-β-dependent and -independent synthesis of connective tissue growth factor (CTGF/CCN2) in liver parenchymal cells in vitro and in vivo. CTGF plays a crucial role in the fibrotic remodeling of various organs which has therefore frequently been proposed as therapeutic target in the management of fibrotic disorders. This article summarizes the clinical-epidemiological observations as well as the pathophysiological background of the antifibrotic effects of coffee consumption and provides suggestions for the therapeutic use of caffeine and its derived metabolic methylxanthines as potentially powerful drugs in patients with chronic fibrogenic liver disease by their inhibitory effect on (hepatocellular) CTGF synthesis.

Full text

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Environmental Toxicology and Pharmacology 28 (2009) 1–10
Contents lists available at ScienceDirect
Environmental Toxicology and Pharmacology
journal homepage: www.elsevier.com/locate/etap
Review
About coffee, cappuccino and connective tissue growth factor—Or how
to protect your liver!?
Olav A. Gressner∗
Institute of Clinical Chemistry and Pathobiochemistry, Central Laboratory, RWTH-University Hospital, Pauwelsstraße 30, 52074 Aachen, Germany
a r t i c l ei n f o
Article history:
Received 30 September 2008
Received in revised form 5 February 2009
Accepted 11 February 2009
Available online 21 February 2009
Keywords:
Coffee
Caffeine
Paraxanthine
CTGF
TGF-?
Smad2
Smad3
PPAR?
Liver fibrosis
a b s t r a c t
Several epidemiological studies suggest that coffee drinking is inversely correlated with the risk of devel-
opment of liver fibrosis. However, a causal, mechanistic explanation has long been pending. New results
indicate that the methylxanthine caffeine, major component of coffee and the most widely consumed
pharmacologically active substance in the world, might be responsible for this phenomenon as it, and
even more potently its derived primary metabolite paraxanthine, inhibits transforming growth factor
(TGF)-?-dependent and -independent synthesis of connective tissue growth factor (CTGF/CCN2) in liver
parenchymal cells in vitro and in vivo. CTGF plays a crucial role in the fibrotic remodeling of various
organs which has therefore frequently been proposed as therapeutic target in the management of fibrotic
disorders.
This article summarizes the clinical–epidemiological observations as well as the pathophysiological
background of the antifibrotic effects of coffee consumption and provides suggestions for the ther-
apeutic use of caffeine and its derived metabolic methylxanthines as potentially powerful drugs in
patients with chronic fibrogenic liver disease by their inhibitory effect on (hepatocellular) CTGF syn-
thesis.
© 2009 Elsevier B.V. All rights reserved.
Contents
1.
2.
3.
4.
5.
6.
Epidemiological studies................................................................................................................................
Caffeine: what is it all about?..........................................................................................................................
Connective tissue growth factor (CTGF/CCN2): structure and protein family ........................................................................
Modulation of CTGF expression by the TGF-? superfamily of cytokines..............................................................................
The role of CTGF in epithelial to mesenchymal transition.............................................................................................
Caffeine suppresses TGF-?-dependent and -independent CTGF synthesis of the hepatocyte primarily through an inhibition of the
TGF-? effector Smad2 .................................................................................................................................
Caffeine upregulates the expression of the nuclear peroxisome proliferator-activated receptor ? in hepatocytes ..................................
Intraperitoneal application of caffeine prevents d-galactosamine induced hepatic expression of CTGF.............................................
Identification of paraxanthine as the most potent caffeine-derived inhibitor of connective tissue growth factor expression in
liver parenchymal cells.................................................................................................................................
Summary and future perspectives....................................................................................................................
Conflict of interest .....................................................................................................................................
References..............................................................................................................................................
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1. Epidemiological studies
“A coffee with your brandy, Sir?”. This citation does not only
reflectEnglishclubtraditions–muchmore,recentscientificreports
proposedamedicalandmolecular-biologicalrationalebehindsuch
cultural habits.
∗Tel.: +49 241 8088671; fax: +49 241 8082512.
E-mail address: ogressner@ukaachen.de.
Liver fibrosis, most commonly caused by alcoholism and hep-
atitis C, and characterized by replacement of functional liver tissue
by fibrotic scar tissue as well as regenerative nodules, was the
11th leading cause of death in the United States in 2001, killing
about 27,000 people each year with a 10-year mortality of 34–66%
(Anderson and Smith, 2003). Disease progression and fibrogenic
activity show significant inter-individual variability, allowing dis-
crimination between slow, intermediate and rapid fibrosers. Both,
environmental and host genetic factors are suspected to mod-
1382-6689/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.etap.2009.02.005

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O.A. Gressner / Environmental Toxicology and Pharmacology 28 (2009) 1–10
ify disease susceptibility and progression rate (Gressner et al.,
2008a).
Data on 5994 adult patients with chronic liver disease,
collected by US-American scientists during the third National
Health And Nutrition Examination Survey (NHANES III) (Ruhl
and Everhart, 2005a,b) of the National Centers of Health Statis-
tics, Disease Control and Prevention (CDC; Atlanta/GA, USA)
proposed a hepatoprotective effect of increased coffee con-
sumption. Similar results were obtained previously by NHANES
I as well as during a recent study by the National Institute
of Diabetes and Digestive and Kidney Disease/National Insti-
tute of Health (NIDDK/NIH; Bethesda/MD, USA) (Modi et al.,
2007).
In summary, these studies gave evidence that patients with
higher coffee consumption displayed a milder course of fibrosis
(Ruhl and Everhart, 2005b; Modi et al., 2007), especially in alco-
holic liver disease (Ruhl and Everhart, 2005a,b; Tanaka et al., 1998)
and lower serum activities of alanin-aminotransferase (ALT) and ?-
glutamyltransferase (GGT) (Ruhl and Everhart, 2005a; Tanaka et al.,
1998). According to Ruhl et al., two cups of coffee daily were suffi-
cient, to markedly reduce the risk of fibrosis progression (Ruhl and
Everhart, 2005b).
Scientists from Tohoku University Hospital in Sendai/Japan who
evaluated 9-year data of coffee consumption of 60,107 subjects for
the association of coffee intake and the risk of developing primary
liver cancer (hepatocellular carcinoma; HCC) found that regular
coffeedrinkershadariskforsufferingfromHCC,whichwassignifi-
cantlyreducedcomparedtothosewhoremainedabstinenttowards
coffee consumption (Shimazu et al., 2005).
The Japan Collaborative Cohort Study for Evaluation of Cancer
Risk(JACCStudy)investigated110,688cohortmembersaged40–79
years in respect of their average coffee intake and calculated a haz-
ard ratio of 0.50 for death due to HCC for drinkers of one and
more cups of coffee per day. In contrast, the ratio for drinkers of
less than one cup per day was 0.83, which therefore confirmed an
inverseassociationbetweencoffeeconsumptionandHCCmortality
(Kurozawa et al., 2005).
These findings were supplemented with a Swedish meta-
analysis of the Karolinska Institute, Stockholm which evaluated
the data of 9 cohort and case–control studies involving a total of
2260 cases and 239,146 non-cases. All epidemiological studies that
were considered reported an inverse relation between coffee con-
sumption and risk of liver cancer, and in 6 studies the association
was statistically significant. Overall, this meta-analysis revealed an
association of an increase in consumption of 2 cups of coffee per
day and a 43% reduced risk of developing HCC (Larsson and Wolk,
2007).
However, despite of these striking epidemiological data, the
cellular and molecular mechanisms underlying the antifibrotic
and tumor-suppressive effects of coffee consumption remained
obscure.
2. Caffeine: what is it all about?
Most of the world’s coffee today comes from either South Amer-
ica or Indonesia, but coffee originated in the highlands of Ethiopia
and did not reach Europe for thousands of years (Weinberg and
Bealer, 2000). It was not until the 16th century that the introduc-
tion of coffee to Europe took place. Coffee arrived from the Middle
East where it had achieved a near cult like following, with the first
coffeehouses being established in Istanbul (Weinberg and Bealer,
2000). Once accepted by Islamic law (it was very nearly banned,
likealcohol)thebeveragefollowedthespreadofIslamacrossAfrica
and Eastern Europe. Venice, which relied heavily upon trade with
the Muslim east, was first introduced to the invigorating liquid
in the 1570s (Weinberg and Bealer, 2000). However coffee would
remain a luxury item at this time and not drunk for refreshment,
but as a medicinal drink. However once coffee was transplanted
to European colonies in Asia and South America the bean thrived
and became accessible to the public (Weinberg and Bealer, 2000).
The17thcenturysawtheopeningofthefirstEuropeancoffeehouse
in Venice, which later spawned over two hundred others along its
canals. Coffee spread quickly at this point and other coffeehouses
were founded in the major cities of Italy. Some of these dignified
and elegant establishments are still in existence in Venice, Turin
and Rome; virtual palaces to the national stimulant (Weinberg and
Bealer, 2000).
AtthebehestoftheGermanwriterJohannWolfgangvonGoethe,
Friedlieb Ferdinand Runge, a chemist and pharmacist from Wro-
claw, was the first to investigate coffee beans with the objective
of finding the psychoactive substance in coffee (Weinberg and
Bealer, 2000). In 1820, he finally extracted chemically pure caf-
feine, from which the German scientists Christoph Heinrich Pfaff
and Justus von Liebig successfully deduced the structural formula
C8H10N4O2by burning analysis (Weinberg and Bealer, 2000). In his
1875 professorial dissertation, the Würzburg chemist and pharma-
cist Ludwig Medicus transferred this structural formula into the
chemical structure 1,3,7-trimethylxanthine (Weinberg and Bealer,
2000). However, after major disputes with Hermann Emil Fischer,
based in Berlin and Nobel Prize winner in 1902, Ludwig Medicus’
chemical structure only received public acceptance after the first
chemical synthesis of caffeine by Fischer in 1895 (Weinberg and
Bealer, 2000).
Today, global consumption of caffeine has been estimated at
120,000tonnes per annum, making it the most widely consumed
pharmacologically active substance in the world (Weinberg and
Bealer, 2000). In North America, 90% of adults consume caffeine
daily (Weinberg and Bealer, 2000). It is completely absorbed by
the stomach and small intestine within 45min of ingestion, and is
eliminated by first-order kinetics (Newton et al., 1981).
Caffeine is metabolized in the liver, particularly in liver
parenchymalcells(hepatocytes),
oxidase enzyme system (CYP1A2) into the three metabolic
dimethylxanthines paraxanthine (1,7-dimethylxanthine; 84%),
theobromine (3,7-dimethylxanthine; 12%), and theophylline (1,3-
dimethylxanthine; 4%) (Barone and Roberts, 1996; Gates and
Miners, 1999; Ullrich et al., 1992; Roberts et al., 1994) (Fig. 1).
Furtherdemethylationandoxidationformuratesanduracilderiva-
tives. About a dozen metabolites can be recovered in the urine of
regular coffee consumers (Barone and Roberts, 1996; Ullrich et al.,
1992).
Caffeine and its metabolites act through multiple mechanisms
involving both action on receptors and channels on the cell mem-
brane,aswellasintracellularactiononcalciumandcAMPpathways
(Nguyen et al., 2007). Even though the major caffeine derivatives,
i.e. paraxanthine, theobromine, and theophylline, have common
mechanisms of action, the fraction, by which any of the pathways
is affected, differs between them.
By virtue of its purine structure, caffeine can act on some of
the same targets as adenosine related nucleosides and nucleotides,
i.e. activation of intracellular Ryanodine receptors [which are the
physiological target of cADPR (cyclic ADP ribose)] in vitro as well
as competitive inhibition of adenosine receptors (particularly to
subtypes 1 and A2) and of the cyclic adenosine monophosphate
phosphodiesterase (cAMP-PDE) in vitro and in vivo (Belibi et al.,
2002; Jafari and Rabbani, 2004; Fisone et al., 2004; Daly, 1993;
Eteng et al., 1997). Inhibition of the latter, in particular, results in an
accumulation of cAMP within the cell.
cAMP was one of the first identified second messengers trans-
mitting signals via G-protein coupled receptors and protein kinase
A (PKA) from the cell surface to the nucleus (Montminy, 1997).
by thecytochromeP450

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Fig. 1. Hepatocellular metabolization of caffeineby the cytochrome P450 oxidase enzyme system (CYP1A2) into the three metabolic dimethylxanthines paraxanthine (1,7-
dimethylxanthine), theobromine (3,7-dimethylxanthine), and theophylline (1,3-dimethylxanthine). In 3D shapes, atoms are color coded: C=grey, N=dark blue, O=red,
H=white.
In the unactivated state, PKA resides in the cytoplasm. Induction
by cAMP liberates the catalytic subunits of PKA, which then are
capable of diffusing into the nucleus where they phosphorylate
transcription factors, i.e. cAMP response element binding protein
(CREB) (Sassone-Corsi, 1998). PKA phosphorylates CREB at serine
133, which then transactivates cAMP-responsive genes by bind-
ing as a dimer to a conserved, 8bp, palindromic cAMP response
element (CRE), TGACGTCA. Over 100 genes with functional CREs
have been identified so far and a modulation of various cell sig-
naling proteins by cAMP has been reported (Mayr and Montminy,
2001).
3. Connective tissue growth factor (CTGF/CCN2): structure
and protein family
We just concluded that caffeine and its primary metabolites
act as competitive intracellular inhibitors of cAMP-PDE, which
converts cAMP to its non-cyclic form (Jeon et al., 2005), thus
allowing cAMP to build up in cells. This aspect is of particular
relevance, as cAMP was shown to inhibit transforming growth fac-
tor (TGF)-? induced connective tissue growth factor (CTGF/CCN2)
expression (Heusinger-Ribeiro et al., 2001; Kothapalli et al., 1998).
CTGF, a 36–38kDa cysteine-rich, heparin-binding, and secreted
protein synthesized by various cell types, is now classified as the
second of six members of the CCN gene family containing CTGF
itself, cyr61, NOV, and others (Rachfal and Brigstock, 2003), which
share approximately 40–60% sequence similarity and are charac-
terized as mosaic proteins that comprise four conserved structural
modules (Leask and Abraham, 2006). These modules are impor-
tant for the pleiotropic functions of CTGF including among others
matrix production, cell migration, cell adhesion, and cellular dif-
ferentiation (Moussad and Brigstock, 2000; Rachfal and Brigstock,
2005).
As may be deduced from these effects, CTGF has reached con-
siderable pathophysiological relevance because of its involvement
in the pathogenesis of fibrotic diseases, carcinogenesis, atheroscle-
rosis, skin scarring, and other conditions with excess production of
connective tissue (Fig. 2) (Rachfal and Brigstock, 2005).
4. Modulation of CTGF expression by the TGF-? superfamily
of cytokines
Recently,theabilityofhepatocytesforthesynthesisofCTGFwas
shown by detailed cell culture studies, which clearly demonstrate
CTGF expression in parenchymal liver cells, and that it is sensitively
up-regulated by exogenous Transforming Growth Factor (TGF-?)
(Fig. 2) (Gressner et al., 2007; Weng et al., 2007). In addition, hepa-
tocellularCTGFexpressionalsooccursspontaneouslyinTGF-?-free
culture conditions due to intracellular activation of latent TGF-?
(Gressner et al., 2008b). Thus, hepatocytes are now recognized as a
quantitatively important source of CTGF, which responds to TGF-?.
TGF-? belongs to a superfamily of cytokines, which comprises
further ligands, such as bone morphogenetic proteins (BMPs),
and Activin A. All TGF-? superfamily ligands bind to a type II
receptor dimer, which recruits a type I receptor dimer form-
ing a hetero-tetrameric complex with the ligand (Gressner et al.,
2002), resulting in the phosphorylation of the type I receptor. The
activated type I receptor then phosphorylates receptor-regulated
SMADs (R-SMADs), which can now bind the coSMAD SMAD4. R-
SMAD/coSMAD complexes accumulate in the nucleus where they
actastranscriptionfactorsandparticipateintheregulationoftarget
gene expression (Gressner et al., 2002).
CTGF gene activation by TGF-? is mediated by a functional
Smad-binding element, which resides within the CCN2 promoter
(Rachfal and Brigstock, 2005). In hepatocytes TGF-?-driven CTGF
gene expression is primarily controlled by Smad2 and its transcrip-
tional cofactors and, to a much lesser extent, by Smad3 (Gressner
et al., 2009), which confirms data obtained in other cellular sys-
tems(Gressneretal.,2008b,c).AlargenumberofSmad2-associated
transcriptional co-activators, including CREB binding protein (CBP)
andp300,havebeenidentifiedtopossessintrinsicacetyltransferase
activities that are important for their abilities to enhance transcrip-

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O.A. Gressner / Environmental Toxicology and Pharmacology 28 (2009) 1–10
Fig.2. Transforminggrowthfactor-?(TGF-?)isimplicatedasakeymediatorinthedevelopmentoforganfibrosis.TherelationshipbetweenTGF-?andCTGFinthestimulation
ofextracellularmatrixsynthesishasbeencharacterizedrecently(MoussadandBrigstock,2000).Basedontheseobservations,ithasbeenproposedthatCTGFisadownstream
mediator of the fibrogenic effect of TGF-?, being implicated in the pathogenesis of wound healing, scleroderma and other fibrotic processes.
tion by this downstream mediator of TGF-? actions (Bannister and
Miska, 2000; Brown et al., 2000; Chen et al., 2001; Kouzarides,
2000; Marmorstein, 2001; Marmorstein and Roth, 2001; Ogryzko,
2001;Rothetal.,2001).InparticularDNAbindingactivityandasso-
ciation with target promoters of Smad2 are tightly regulated by
CBP/p300-mediated acetylation of this Smad in response to TGF-?
signaling (Simonsson et al., 2006).
5. The role of CTGF in epithelial to mesenchymal transition
The fibrogenic mechanisms in the liver are dependent on an
interplay of many pro- and anti-fibrotic/-inflammatory cytokines
(Gressner, 1996; Pinzani and Rombouts, 2004). The hierarchy of
pro-fibrogenic growth factors most importantly includes TGF-?,
designated as “fibrogenic master cytokine” with multiple effects
onextracellularmatrixturnover(Gressneretal.,2002;Bisselletal.,
2001), hepatocellular apoptosis (Yang et al., 2006; Oberhammer et
al., 1991; Gressner et al., 1997, 1996), proliferation and liver regen-
eration (Bissell et al., 2001; Inagaki and Okazaki, 2007; Huang and
Huang, 2005), inflammation and immunosuppression (Cerwenka
and Swain, 1999), and cancerogenesis (Elliott and Blobe, 2005). The
naturalantagonistofmanyactionsofTGF-?isbone-morphogenetic
protein 7 (BMP-7), a member of the TGF-? superfamily (Chen et
al., 2004). Thus, the balance of both growth factors, i.e. TGF-?
and BMP-7, will be crucial for development of fibrosis and out-
come of (chronic) liver disease, i.e. risk for the development of
HCC.
Even though the molecular mechanism of action of CTGF is still
not known in detail yet, a modulator role in the epithelial to mes-
enchymal transition (EMT) of adhering hepatocytes into cells with
reduced intercellular adhesion, increased motility and mesenchy-
mal,fibroblast-likeproperties,isdiscussed(Abreuetal.,2002).This
process is gaining more and more importance in the pathogenetic
understanding of hepatic fibrogenesis (Zeisberg et al., 2003, 2007),
but accumulating evidence also points to a critical role of EMT-like
events during tumor progression and malignant transformation,
endowing the incipient cancer cell with invasive and metastatic
properties (Larue and Bellacosa, 2005).
The prototype of the currently most powerful inducer of EMT
is TGF-? (Zavadil et al., 2004), activating this pathway via induc-
tion of Smad2/3 phosphorylation and the Snail transcription factor
(Zavadiletal.,2004).Incontrast,BMP-7,themostimportantmolec-
ular counterpart of TGF-?, not only inhibits EMT, but can even
induce a mesenchymal–epithelial transition (reverse EMT=MET)
(Zeisberg et al., 2003). Recent reports gave evidence that up-
regulation of CTGF inhibits BMP-7 signal transduction in the
diabetic kidney (Nguyen et al., 2008). Abreu et al. furthermore
presented data describing CTGF as extracellular trapping protein
for BMP and TGF-? (Abreu et al., 2002). According to their func-
tionalstudiesonXenopuslaevis,CTGFdirectlybindsBMPandTGF-?
through their cysteine-rich (CR) domain, thus antagonizing BMP
activity by preventing its binding to BMP receptors. Of note, the
oppositeeffect,enhancementofreceptorbinding,wasobservedfor
TGF-? (Abreu et al., 2002). These results suggest that CTGF inhibits
BMP and activates TGF-? signals by direct binding in the extracel-
lular space. From this, CTGF would act pro-fibrogenic (Fig. 3).
A central role of CTGF in liver fibrogenesis and tumor growth,
whichmaythusbeexpected,isdocumentedbyreportsonincreased
CTGF expression in various tumor tissues (Gressner and Gressner,
2008; Liu et al., 2008; Mullis et al., 2008; Munemasa et al., 2007;
Boag et al., 2007; Kidd et al., 2007) as well as in fibrotic liver
tissue (Fig. 4) (Rachfal and Brigstock, 2003; Hayashi et al., 2002;
Paradis et al., 1999), and, even more important, by recent studies,
in which knock-down of CTGF by siRNA leads to substantial attenu-
ation of experimental liver fibrosis (George and Tsutsumi, 2007; Li
et al., 2006). Thus, modulators of CTGF-expression will have a great
pathogenetic relevance for fibrosis.
6. Caffeine suppresses TGF-?-dependent and -independent
CTGF synthesis of the hepatocyte primarily through an
inhibition of the TGF-? effector Smad2
We previously talked about Smad2 (and, to a lesser extent,
Smad3) as key mediator of TGF-?-induced CTGF expression in hep-
atocytes (Gressner et al., 2009).
Very recent observations gave evidence, that caffeine is able to
enforce proteasomal Smad2 degradation in hepatocytes (Fig. 6) by

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Fig. 3. The impact of connective tissue growth factor (CTGF/CCN2) on hepatocellular epithelial-to-mesenchymal transition during hepatic fibrogenesis. Hepatocytes are
induced to undergo epithelial-to-mesenchymal transition (EMT) or apoptosis. The resulting fibroblast-like cells lose their ability to express albumin while they become
positive for the fibroblast-specific protein-1 (FSP1). In all these processes, TGF-? acts as a strong inductor of EMT while BMPs have an opposing effect. The balance of both
cytokines is modulated by CTGF that increases TGF-? and reduces BMP activities.
enhancing, the activity of SMURF2, a member of the family of E3
ubiquitin ligases (Lo and Massague, 1999; Zhang et al., 2001), with
the consequence that Smad2 is increasingly bound to ubiquitin and
proteasomally degraded (Gressner et al., 2008d; Lo and Massague,
1999; Zhang et al., 2001; Wicks et al., 2006). This finding seems
to be of particular relevance for clinical situations of TGF-? acti-
vation such as viral hepatitis and tumor growth (Gressner et al.,
2002; Elliott and Blobe, 2005; Massague et al., 2000; Murawaki et
al., 1998), as degradation of Smad2 in response to TGF-? requires
receptor-mediated phosphorylation of the C-terminal serines (Lo
and Massague, 1999; Zhang et al., 2001), which would suggest a
normal or even stimulated Alk5-dependent phosphorylation, i.e.
intra- or extracellular presence of TGF-? (Gressner et al., 2008b).
Enhanced degradation of Smad3 was not observed in the pres-
ence of caffeine, but an inhibition of its phosphorylation (Fig. 6),
which indicates a stimulation of proteasome-mediated degrada-
tion, specifically of Smad2, by caffeine. Such a high degree of
specificity of SMURF2 to preferentially degrade Smad1 and Smad2
but not Smad3, was previously described by Lo et al. (Lo and
Massague, 1999; Zhang et al., 2001). The caffeine-induced inhibi-
tion of phosphorylation of Smad3, however, may still be secondary
to enhancement of SMURF2 activity, as this ubiquitin ligase is also
Fig.4. ImmunohistochemicalstainingofCTGFinnormalandbile-ductligatedfibroticratliver(Gressneretal.,2008).LocalizationofCTGFincytokeratin18positivehepatocytes
and in few desmin-positive (myo-)fibroblasts is shown.