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					TGF – role in osteoarthritis. A review
Alejandro Melo–Florián MD
Internal Medicine Specialist - Pontificia Universidad Javeriana, Bogotá D.C.
Corresponding author: Alejandro Melo-Florián
Email address: alejandromeloflorian@gmail.com

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Como citar:
Melo-Florián, Alejandro. TGF-β role in osteoarthritis. A review
[Internet]. Version 8. Open Journal of Medicine. 2011 Sep 8.
Available       from:     http://knol.google.com/k/alejandro-melo-
florián/tgf-β-role-in-osteoarthritis-a-review/3sktw3ldc86j2/189.

ABSTRACT:
Cytokines role in osteoarthritis is now well recognized and impact the normal
function or homeostasis of hyaline articular cartilage. TGF- altogether with
another cytokines such as BMP are regulatory or anabolic cytokines interact
with the chondrocyte in a paracrine pathaway. TGF-β usually signals through a
pair of transmembrane serine/threonine kinases known as the type I and type II
TGF-β receptors and the deregulation of its signalling is implicated in OA. Once
TGF-β ligand bind to specific type II receptors, the Smad-dependent TGF-β
signaling pathways initiate a cascade of events yielding to phosphorylation of
their specific receptor-Smads (R-Smads); TGF-β signaling depends on Smad2
and Smad3 proteins. As a pharmacological target, the stimulation of TGF-
expression in articular chondrocytes by diacerhein, further to its inhibition of IL-
1, could favour cartilage homeostasis promoting anabolic processes in the OA
cartilage, therefore contributing to delay the progression of the disease. In
conclusion, drugs acting in chondrocyte in the pathway of TGF-β could favour
anabolic processes in the OA cartilage, contributing to delay the progression of
the disease, and adjusting to the definition of disease-modifying osteoarthritic
drug which interferes with the cartilage breakdown observed and improve
symptoms.

Key words: Cartilage homeostasis; Osteoarthritis; TGF-β; Anabolic state.




TGF – role in osteoarthritis. A review. Alejandro Melo Florián MD
Abreviatures: – NO Nytric Oxide – OA: Osteoarthritis – TGF-: Transforming
Growth Factor-beta – TIMP: tissue inhibitor of metalloprotease – Smad proteins:
similar proteins Drosophila, mothers against (Mad) –

TGF- and normal cartilage homeostasis
Progressive cartilage degradation is considered a hallmark of osteoarthritis
(OA), and methods to inhibit this process have been extensively investigated as
potential disease-modifying therapies. The new concepts in OA pathogenesis
pinpoints to an altered state of homeostasis, namely the desequilibrium
between anabolic (TGF-, Bone Morphogenetic Protein-BMP) and catabolic
cytokines (IL-1, TNF, IL-6) acting on chondrocyte-dependent synthesis of
cartilaginous matrix finally affects all tissues of the joint in OA, and it is likely
that the pain and disability which are the major clinical symptoms of OA, arise
predominantly from pathology in these extra-cartilaginous structures.
Homeostasis of the extracellular matrix (ECM) of articular cartilage is dependent
on the responses of articular cartilage cells to auto- and paracrine anabolic and
catabolic pathways (Verbruggen G, 2006).
Chondrocytes are the active component present in cartilage, responsible for
maintaining the low turnover state of the synthesis and replacement of the
different components of the matrix, namely collagen and proteoglycan
aggregates. In cartilage, the homeostasis or equilibrium can be described as an
equal rate of deposition of de novo synthesized collagens and proteoglycans
replacing disrupted and damaged collagens and proteoglycans (Goldring MB &
Marcu KB, 2009).
Synthesis and accumulation of the extra-cellular matrix is regulated by locally
produced growth factors, such as the insulin-like growth factors (IGFs) and
transforming growth factor- β (Verbruggen G, 2006). TGF-β is a regulatory or
anabolic cytoquine which plays a critical role in cartilage homeostasis. Normally,
TGF-β isoforms signal through a pair of transmembrane serine/threonine
kinases known as the type I and type II TGF-β receptors and the deregulation of
its signalling is implicated in osteoarthritis (OA) (Finson KW et al, 2010; Parker
WL et al, 2007). Further, TGF-β stimulates collagens and proteoglycans
synthesis by chondrocyte, and reduces the activity of IL-1β stimulated
metalloproteases (Finson KW et al., 2010).

Normal physiology of cartilaginous matrix components
Taking into account the basic purpose of cartilage to provide a suitable covering
material for the articular end of the bones at synovial joints, the covering
material does not thin as a result of the physiological joint activity and this
resistance is due to the nature of the components, able to hold resistance.
Compressive resistance is bestowed by the large proteoglycan aggregates, and
the its main molecule, the aggrecan, which is attached to hyaluronic acid
polymers via link protein. The half-life of aggrecan core protein ranges from 3 to
24 years, and the glycosaminoglycan components of aggrecan are synthesized
more readily under low-turnover conditions. The proteoglycan aggregates are
essential for protecting the collagen network, which has a half-life of more than
100 years if not subjected to inappropriate degradation (Goldring MB & Marcu
KB, 2009). Chondrocytes are the only cells of articular cartilage, and they are
responsible for the production of the extracellular matrix. Chondrocytes are
distributed throughout the matrix and compose less than 5% wet weight;


TGF – role in osteoarthritis. A review. Alejandro Melo Florián MD
collagen makes up about 15-22% of the wet weight and contains 90-95% type II
collagen fibers with a small percentage of types IX and XI (Melero-Martin JM &
Al-Rubeai M, 2007).

Altered cartilaginous homeostasis during OA
In general, catabolic cytokines such as IL-1 are known to cause a decrease in
the expression of cartilage-specific collagens, proteoglycans and TIMPs, while
concomitantly causing increases in the expression of matrix metalloproteinases,
cyclooxygenases and NO. During OA, proteoglycan loss and type II collagen
cleavage initially occur at the cartilage surface, with evidence of pericellular
damage in deeper zones as the lesion progresses (Goldring MB & Marcu KB,
2009).
The turnover and degradation of the extracellular matrix depend on the
responsiveness of the articular cartilage cell to catabolic cytokines, of which IL-
1 and IL-1 are the main agonists (Verburggen G, 2006).

TGF- and normal physiology of chondrocytes
In hyaline cartilage healthy chondrocytes remain in a postmitotic quiescent state
throughout life, with their decreasing proliferative potential being attributed to
replicative senescence associated with erosion of telomere length degradation
(Goldring MB & Marcu KB, 2009).
Bernstein et al., (Bernstein P et al., 2010) used immunohistology and
quantitative reverse transcribing PCR (RT-PCR) to evaluate chondrogenic-
specific markers (i.e., Sox9, Collagen II). They found that Global gene
expression of the so-cultivated chondrocytes with separate samples in
transforming growth factor (TGF)β+ or TGFβ- conditions by use of quantitative
real-time PCR (RT-PCR), resulted in chondrogenic transformation of the
cultured cells.
Interestingly, chondrocytes demonstrated an upregulated fatty acid/cholesterol
metabolism which give interesting tips for optimization of culture conditions.
In culture, isolated chondrocytes steadily lose the expression of the cartilage-
specific genes such as enconding collagens and proteoglycans. However, the
loss of chondrocyte differentiated characteristics in vitro has been linked to the
reduced expression of Sox-9, one of the specific transcription factors (Kolettas
E et al., 2001).
Sox9 is an HMG-box-containing transcription factor, which binds and activates
this enhancer element, is necessary for chondrocyte differentiation and for
expression of a series of chondrocyte-specific marker genes including Col2a1,
Col9a2, Col11a2 and Aggrecan (de Combrugee B, 2000)

Mecanism of action of TGF-β in human chondrocytes
The work of Parker et al, done in human chondrocyte cell lines C28/I2 and
tsT/AC62, in human cartilage tissue specimens, and human primary
chondrocytes determined the expression of receptor type TRII-B (a splice
variant of the type II TGF- receptor) by Western blot and reverse-transcriptase-
polymerase chain reaction. Further, the regulation of TGF- responses by
TRII-B was determined by examining Smad2 phosphorylation, Smad3-specific
signaling, transcriptional activity, and type II collagen levels.




TGF – role in osteoarthritis. A review. Alejandro Melo Florián MD
The results were that TRII-B is expressed in normal as in osteoarthritic human
cartilage, forming heteromeric complexes with the types I and II TGF-beta
receptors (Parker WL et al, 2007)
In the work of Roman-Blas et al. (Roman-Blas JA et al, 2007), they work with
human articular chondrocytes isolated from knee joints from patients with
osteoarthritis (OA) or normal bovine chondrocytes and cultured it in suspension
in polyHEMA-coated dishes with either 10% FBS media or serum-deprived
media six hours before treatment with cytokines, namely: IL-1β alone, TNF-α
alone or IL-1β followed by TGF-β. The results were that pre-treatment with IL-
1β in human OA and bovine chondrocytes reduced TGF-β-induced Smad3/4
DNA-binding activity and Smad2/3 phosphorylation, depicting an antagonic
relationship.
In another hand, real-time PCR and Western blot analysis showed that IL-1β
partially reversed the TGF-β stimulation of Smad7 mRNA and protein levels in
TGF-β-treated human OA cells. In contrast, TGF-β-stimulated COL2A1,
aggrecan, and the chondrogenic marker SOX9 mRNA levels. It means IL-1
exerted a suppressive effect on Smad3/4 DNA-binding activity in human
articular chondrocytes, as well as on TGF- -induced stimulation of Smad3/4
DNA-binding activity and Smad2/3 phosphorylation in human OA and bovine
articular chondrocytes.
The work of Qiao et al. (Qiao B et al., 2005) using rabbit articular chondrocytes,
it demonstrated an enhanced synthesis of type II collagen protein by TGF- and
BMP2, through the way of protein kinases, specifically TGF--activated kinase 1
(TAK1).
In conclusion, growth factors like TGF- during chondrocyte expansion not only
influence cell proliferation and differentiation, but also the influence the cell
potential to redifferentiate and respond to regulatory molecules. TGF-
regulates a large variety of cellular activities and the binding of TGF- to its cell
surface receptor triggers several signaling cascades, among which the TGF--
Smad pathway is the most extensively studied. TGF- also activates protein
kinases, including MAPK, PKA and PKC, and modulates gene expression via its
delicate interaction with other signaling pathways (Li TF et al., 2005)

What are the Smads?
Smad is the acronym corresponding to homologue or similar proteins to protein
called      Drosophila,      mothers    against    (Mad).     In     vertebrates,
five Mad and Sma homologues have thus far been identified and termed 'Smad'
genes (Derynck R and Zhang Y, 1996).
Smad family members are essential intracellular signalling components of the
transforming growth factor- (TGF-) superfamily. Smad2 and Smad3 are
structurally highly similar and mediate TGF- action in chondrocyte as well as in
other cells. In general, activation of TGF- superfamily serine/threonine kinase
receptors leads to the phosphorylation of R-Smads (Smads1, 2, 3, 5, and 8).
This is followed by recruitment of the Co-Smad (Smad-4) into an R-Smad/Co-
Smad complex that translocates to the nucleus to regulate gene expression
(Nakao A et al, 1997)
In the way Smads are physiological signaling components of TGF-, the
catabolic cytokines downregulates DNA-binding activity. According to the work
of Roman-Blas et al. analyzing by electrophoretic mobility-shift assays (EMSA)


TGF – role in osteoarthritis. A review. Alejandro Melo Florián MD
found down-regulation of 42% of DNA binding activity of Smad3/4 in the
TranSignal Protein/DNA array with IL-1β treatment (Roman-Blas JA et al.,
2007).

TGF-  and its actions on chondrogenic fenotype of chondrocyte
The rate of chondrocyte maturation is tightly regulated through the interactions
of TGF--Smad-mediated signaling, the Wnt signaling pathway, and the
transcription factor Runx2.
According to the work of Wang et al. (Wang J et al., 2003), when analyzing
paired normal and OA chondrocytes from the same knee joint, it has been
shown an enhanced capacity of chondrocytes from OA cartilage to produce
extracellular matrix macromolecules. However, the OA cells have increased
catabolic signalling pathways. As a consequence of this increased IL-1 activity
and the reduced amounts of IL-1RII decoy receptor, less of the produced
extracellular matrix macromolecules may persist in the area around the OA
chondrocytes. These data suggest that TGF- / Smad3 signals are essential for
repressing any articular chondrocyte differentiation. Without these inhibiting
signals, chondrocytes break their quiescent state and undergo abnormal
terminal differentiation, leading to osteoarthritis.
Unlike most cartilage, articular cartilage is arrested before terminal hypertrophic
differentiation. According to the work of Yang et al, (Yang X, et al., 2001) TGF
/Smad3 signals inhibit terminal hypertrophic differentiation of chondrocyte and
are essential for maintaining normal articular cartilage.

TGF-  and anabolic actions in chondrocyte and human cartilage
To date, is clear the concept of homeostasis in hyaline cartilage, and the
balance between the IL-1β and the TGF-β signaling pathways is crucial for
maintenance of articular cartilage homeostasis. On the opposite side, the
disruption between these two cytokines likely plays a substantial role resulting
in the pathogenesis of OA. TGF-β can elicit an increase in aggrecan and
collagen gene expression and also prevent loss of proteoglycan in articular
cartilage during experimental OA. In early phases of OA, there is evidence of
compensatory increases in type II collagen synthesis in deeper regions of OA
cartilage (Goldring MB & Marcu KB, 2009).

TGF receptors in chondrocyte
TGF-β family members are vitally important to the cartilage homeostasis in the
adult tissues, its role is allowed by receptors in the surface of chondrocytes. Of
note, this receptors have variable affinity and response of the chondrocytes.
By using affinity labeling in combination with SDS-PAGE, Glansbeek HL et al.
demonstrated a difference in size of the TGF-beta type II receptor between
freshly isolated and cultured chondrocytes, and this difference explains the
differential effect of TGF- on the proteoglycan synthesis of chondrocytes
cultured for 1 or 14 days. Only the TGF- receptor of the 14 days cultured
chondrocytes stimulates the proteoglycan synthesis (Glansbeek HL et al.,
1993).
Once TGF-β or BMP ligands bind to specific type II receptors, the Smad-
dependent TGF-β and BMP signaling pathways recruit the corresponding type I
receptor in order to initiate a cascade of events yielding to phosphorylation of
their specific receptor-Smads (R-Smads); TGF-β signaling depends on Smad2

TGF – role in osteoarthritis. A review. Alejandro Melo Florián MD
and Smad3 proteins. Afterwards, the phosphorylated R-Smad forms a
heterocomplex with the Smad4, the common partner Smad (CoSmad). The R-
Smad/Co-Smad complex translocates into the nucleus, where it binds to
promoters of target genes and regulates their transcription (Keller B et al., 2011)

Therapeutical implications of TGF- stimulation
TGF- plays a significant role in promoting chondrocyte anabolism in vitro
(enhancing matrix production, cell proliferation, chondrogenic differentiation)
and in vivo (short-term intra-articular injections lead to increased bone formation
and subsequent cartilage formation, beneficial effects on chondrogenesis). In
vivo induction of the expression of TGF- and the use of gene transfer may
provide a new approach for treatment of osteoarthritic lesions (Grimaud E, et
al., 2002).
In another hand, it has been reported that the culture of chondrocytes expanded
in monolayer in the presence of FGF-2/TGF-β, displayed a higher proliferation
rate and more dedifferentiation, but also higher capacity to differentiate in
response to TGF-β and dexamethasone during three-dimensional cultures.
Similar results have been recently reported by culturing bovine
chondroprogenitor cells in the presence of TGF-β1 and this evidence confirms
somehow that pharmacological agents stimulating TGF-β will have a favorable
impact in chondrocyte metabolism (Melero-Martin JM & Al-Rubeai M, 2007).

Pharmacological actions of DMOAD mediated through TGF-
Some agents have evidence of action in OA stimulating TGF-β. The data
indicated that active principles such as diacerein or diacehtylrheine enhance the
expression of TGF-1 and TGF-2. This effect was also found in the presence
of IL-1, albeit with smaller intensity. It seems that the anabolic action of
diacerhein is mediated through the effect of TGF-.
In contrast, the levels of TGF-3 and receptors I and II remained unaffected or
slighty modified by the compound. Treatment of cells transiently transfected
with TGF-1 promoter constructs suggested that the stimulating effect on TGF-
1 expression is mediated by the region -1038 to -1132 base pars. This
mechanism of action may account for the potential disease-modifying properties
of diacerhein and it is worth to take into account given the rising incidence of the
disease (Felisaz N et al, 1999).
By the stimulation of TGF- expression in cultured articular chondrocytes,
further to its inhibition of IL-1, diacerhein could favour cartilage homeostasis
promoting anabolic processes in the OA cartilage, therefore contributing to
delay the progression of the disease (Pujol JP et al, 2000). This effect is a
rational and compatible basis with the definition of the disease-modifying
osteoarthritic drug (DMOAD) which interferes with the cartilage breakdown
observed and improve symptoms or prevent deterioration of the patient's clinical
condition (Dougados M et al., 2009)

Further, diacerhein at therapeutically useful concentrations, consistently inhibits
the catabolic effect of cytokines (such as IL-1, IL-1) on newly secreted
proteins, avoiding the effects of metalloproteinase activity and nitric oxide
production, whereas a selective nitric oxide blocker agent alone is ineffective.
The pattern of protein secretion and metalloproteinase activity in chondrocytes
from elderly subjects seems different from that of young patients, but was highly

TGF – role in osteoarthritis. A review. Alejandro Melo Florián MD
expressed in osteoarthritic chondrocytes. These data consistently demonstrate
that a specific gene program is turned-on in catabolic-cytokine-stimulated
chondrocytes, thus resulting in production of enzymes involved in remodeling
and destruction of cartilage matrix (Dozin B et al, 2002).

Conclusions
The normal structure and function of articular cartilage is the result of a well
timing balanced interaction between anabolic and catabolic processes. The
transforming growth factor-beta (TGF-) family of growth factors exerts an
anabolic or repair response replacing disrupted elements in cartilaginous matrix;
its effects are mediated through different molecular mechanisms, such as the
Smad pathways, the protein kinases pathways (MAPK, PKA and PKC).
In contrast, pro-inflammatory cytokines such as interleukin 1(IL-1) originated
in synovial membrane exerts a strong catabolic effect. Recent evidence has
shown that IL-1, TNF- in a lesser degree, and the TGF- signaling pathways
share an antagonistic relationship.
According to the literature, diacerhein could favour cartilage homeostasis
promoting anabolic processes in the OA cartilage, therefore contributing to
delay the progression of the disease, then fitting to the definition of disease-
modifying osteoarthritic drug which interferes with the cartilage breakdown
observed and improve symptoms.

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Competing interests: Author is working with a pharmaceutical manufacturer promoting diacerhein.




TGF – role in osteoarthritis. A review. Alejandro Melo Florián MD

				
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