chondrogenesis is the earliest phase of skeletal development , involving mesenchymal cell recruitment and migration , condensation of progenitors , and chondrocyte differentiation and maturation .
mutations or deregulation of these processes lead to skeletal malformation , skeletal malfunction , and/or susceptibility to injury .
each step of the chondrocytic differentiation process is characterized by specific morphological change and gene expression profiles .
several cytokines , growth factors , extracellular matrix ( ecm ) components , and transcription factors have important roles in these processes .
two groups of transcription factors have been identified that control the key steps of chondrocyte differentiation : the sox and runx families .
the sox trio of transcription factors ( sox9/sox5/sox6 ) has important regulatory functions in the early stages of chondrogenesis .
sox5 and sox6 are coexpressed with sox9 in pre - chondrocytes and proliferating chondrocytes , and the three sox proteins cooperate with each other to directly activate type ii collagen ( col2a1 ) and aggrecan in vitro .
sox9-null mutant cells in mouse embryo chimera are excluded from mesenchymal condensations and fail to express col2a1 and other chondrocyte - specific markers ( col9a2 , col11a2 , and aggrecan ) , indicating that sox9 is required for mesenchymal condensation and subsequent cartilage formation .
sox5 and sox6 have essential but somewhat redundant roles in promoting chondrogenesis , and double - null mice fetuses die at embryonic day 16.5 ( e16.5 ) from heart failure with severe chondrodysplasia .
runx2 is mainly expressed when chondrocytes are about to become pre - hypertrophic , and it remains expressed through hypertrophy and terminal differentiation .
runx2-null mice have abnormal hypertrophic chondrocyte differentiation in addition to failure of bone formation . however , there is failure of terminal differentiation only in the humerus and femur and delayed terminal differentiation in other long bones , including tibia , fibula , radius , and ulna .
the transcription factor c - maf is specifically expressed in late hypertrophic and terminal chondrocytes , making it a candidate for controlling terminal differentiation .
in addition , the expression of c - maf and lc - maf , an mrna splicing variant of c - maf , partially overlaps with the expression of both sox9 and runx2 .
the founding member of maf family , v - maf ( musculoaponeurotic fibrosarcoma ) , was originally identified as a retroviral oncoprotein , and then cellular counterparts , c - mafs , were cloned from a number of vertebrate genomes .
the maf family is currently subdivided into two groups , small maf ( maff , mafg , mafk , maft , and mafs ) and large maf ( c - maf , mafa , mafb , and nrl [ neural retina leucine zipper ] ) proteins based on their domain structure and function ( table 1 ) . a transcript variant of c - maf , lc - maf , also has been reported ( fig .
the large maf proteins contain a conserved n - terminal transactivation domain , whereas the small maf proteins lack this transactivation domain .
based on the strong sequence conservation , the members of the maf family are classified under the bzip transcription factor superfamily .
binding partners of mafs homo- and heterodimers form between mafs and other bzip family proteins .
c - maf and its rna splice variant lc - maf ( long form c - maf ) contain the same 5 untranslated region .
c - maf is transcribed from a single exon , and through mrna splicing , lc - maf has a frame shift that alters the last amino acid of c - maf exon 1 and codes for an additional 10 amino acids ( in mouse ) or 30 amino acids ( in human ) .
the bzip transcription factor superfamily consists of 53 different proteins in humans and has been subdivided into several subgroups , including the hallmark ap-1 complex ( c - fos / c - jun ) , creb / atf , cnc , c / ebp , par , and maf families .
bzip transcription factors are activated by a variety of signal transduction pathways to mediate critical aspects of cellular differentiation .
the bzip structural motif is a long bipartite -helix of 60 to 80 amino acids in length and comprises a basic region and a leucine zipper .
the n - terminal basic region contains an 18amino acid basic peptide responsible for specific dna recognition , whereas the c - terminal leucine zipper domain mediates homo- and heterodimerization with other bzip proteins .
the maf family members are unique among the bzip superfamily in that they contain a highly conserved ancillary dna binding domain n - terminal to the typical basic domain ( fig .
1 ) , which increases the number of specific dna sequences that can be bound . through this extended dna binding domain ,
maf proteins bind to relatively long consensus dna sequences termed maf - recognition elements ( mares ) .
early in vitro selection studies identified two 13 and 14base pair palindromic sequences , tgctgactcagca ( t - mare ) and tgctgacgtcagca ( c - mare ) , as the optimal binding sites for c - maf .
the t - mare contains a phorbol 12-o - tetradecanoate ( tpa)response element ( tre : tgactca ) , and the c - mare contains a cyclic amp - responsive element ( cre : tgacgtca ) in the middle region , respectively .
however , in vitro studies have shown that some base mismatches from the consensus sequences are allowed in maf binding to the mare and that maf homodimers also bind efficiently to mare half - palindromic sites when they are preceded by an at - rich sequence . naturally occurring c - maf binding sites in il-4 , crystallin , and other genes
are not intact palindromic sequences , as the full - consensus sequences are present only in the 5 half - region .
therefore , although there is a degree of specificity in the maf binding to the target dna sequences , there is also considerable flexibility .
in the developing mouse embryo , c - maf is strongly expressed in the lens fiber cells , and studies have focused on c - maf s function in lens development .
expression of c - maf and mafb was detected at the rna level in limb and rib cartilage as well as eye lens and spinal cord of the e15 rat embryo .
the analysis of postnatal rat femur epiphysis in the same study showed that c - maf is expressed strongly in the hypertrophic chondrocytes but low in immature proliferating chondrocytes . a detailed localization study in mouse embryonic cartilage showed that c - maf is expressed mostly in late hypertrophic and terminally differentiated chondrocytes with a maximal expression at e15.5-e16.5 and at low levels in the perichondrium and in the primary spongiosa . on the other hand , lc - maf
lc - maf expression is detected only in the perichondrium , with no expression in differentiated chondrocytes .
although the differential expression patterns of c - maf and lc - maf during chondrogenesis were described , the functional differences between c - maf and lc - maf are still unknown .
thus , the regulation mechanisms that control temporal - spatial expression of the different c - maf isoforms need to be further investigated .
the prominent phenotype of the c - maf - null mice is defective lens fiber cell differentiation .
direct transactivation of lens - specific crystallin genes by c - maf has been related to this phenotype .
although c - maf ( + / ) heterozygous mice have normal lenses , naturally occurring heterozygous point mutations in c - maf cause mild cataracts in both mice and humans .
these mutations in the basic domain may have a dominant negative effect on wild - type c - maf protein . whether these mutations are related to abnormal cartilage formation
it is interesting to note that lens and cartilage share similar gene expression profiles , especially genes involved in ecm such as collagens , other noncollagenous ecm proteins , and matrix metalloproteinases ( mmps ) .
the role of c - maf in cartilage was examined in endochondral bone development in mice lacking c - maf .
c - maf - null mice have abnormal terminal differentiation of hypertrophic chondrocytes with the most dramatic abnormality at e15.5 to e16.5 , which corresponds to the time points of the highest c - maf expression in chondrocytes .
terminal differentiation of the hypertrophic chondrocytes is delayed , and the domain of late hypertrophic chondrocytes is expanded in both the e16.5 to e18.5 embryos and 4-week postnatal c - maf - null mice . the expanded hypertrophic zone is associated with delayed vascular invasion and reduced ossification .
this suggests that c - maf facilitates both the initiation and the completion of terminal differentiation in chondrocytes .
the maf proteins dimerize with themselves and with other bzip proteins through the leucine zipper domain .
the maf binding partners that have been identified so far are summarized in table 1 , and the various types of c - maf binding interactions are illustrated diagrammatically in figure 2 .
there is specificity in maf dimerization , and the different binding interactions affect the transactivation function of the maf proteins .
however , the circumstances that determine the binding of maf to its partners are not fully understood .
the small maf proteins , which do not dimerize with large mafs , generally repress transcription when they form homodimers .
in contrast , when they form heterodimers with other members of bzip proteins , they can activate or repress transcription of genes regulating many aspects of cellular differentiation .
a heterodimeric partner of small maf , the transcription factor nuclear factor e2 p45-related factor-2 ( nrf2 ) , displays decreased expression during chondrocyte differentiation and inhibits numerous aspects of chondrocyte differentiation in vitro .
this suggests the possibility that small maf / nrf2 heterodimers may be involved in the negative regulation of chondrocyte differentiation , although no expression data for small mafs are available in chondrocytes .
they also have the ability to form heterodimers with ap-1 family members in vitro , although heterodimers have not yet been detected in vivo . 
 in vitro
, c - maf and nrl can form heterodimers with c - fos and c - jun .
mafa can heterodimerize with c - jun but not with c - fos , whereas mafb can form heterodimers with c - fos but not with c - jun . considering that maf proteins are expressed in a wide variety of cells and tissues ,
it has been suggested that mixing and matching of dimeric partners might create specific complexes within cells to affect target gene expression and cell differentiation .
c - maf can form homodimers as well as heterodimers with other bzip transcription factors .
in addition , several factors , including general transcription factors ( tbp ) , cofactors ( cbp ) , and specific transcription factors ( sox9 ) , interact with c - maf .
mafs can also physically and/or functionally interact with several non - bzip proteins , including general transcription factors , common coactivators , and specific transcription factors ( fig .
nrl directly interacts with tata binding protein ( tbp ) in vitro , and tbp is co - immunoprecipitated with the nrl- , c - maf- and mafa - containing complexes in vivo .
c - maf recruits the creb binding protein ( cbp ) and p300 to the promoters of crystallin genes , resulting in the synergistic activation of the genes .
this synergistic activation requires histone acetyltransfease activity , indicating that chromatin remodeling is involved in c - maf transactivation .
synergy in transcriptional activation by c - maf and other specific transcription factors was also observed .
c - maf acts synergistically with sox1 , sox2 , and sox3 , which are important regulators of lens development , to activate the f - crystallin gene . similarly ,
lc - maf acts synergistically with sox9 to activate the col2a1 and type xxvii collagen ( col27a1 ) gene transcription in chondrocytes .
although maf was first identified as a retroviral oncoprotein , the physiological functions of maf family proteins include regulatory roles in a wide variety of cell- and tissue - specific gene expression and in cell differentiation during development .
the target genes of maf transcription factors in tissues are well summarized ( see review by kataoka ) .
this section focuses on c - maf targets in chondrocytes , summarizing various lines of evidence that c - maf regulates genes related to chondrocyte differentiation and the progression of osteoarthritis .
this growing list of genes includes matrix metalloproteinase-13 ( mmp-13 ) , connective tissue growth factor ( ctgf ) , col2a1 , and col27a1 ( fig .
schematics of c - maf target genes in chondrocytes and possible pathways regulating c - maf regulation . fgf / mapk / erk pathway may activate and affect the stability of c - maf , based on homology with interactions affecting mafa .
some interactions are proposed based on homology of the phosphorylation / ubiquitination sites between mafa and c - maf .
these interactions still need to be verified for c - maf , as clarified in the text .
mmp-13 , along with other matrix metalloproteinases , affects hypertrophic differentiation and vascular invasion through matrix proteolysis .
mmp-13 is induced in terminally differentiated chondrocytes in the developing growth plate and is highly upregulated in osteoarthritic cartilage .
mmp-13 expression in the hypertrophic chondrocytes region was decreased specifically in the c - maf - null mice embryo , suggesting a regulatory role for c - maf in mmp-13 expression .
this is supported by a study of c - maf activity on the mmp-13 proximal promoter .
however , there is the possibility that c - maf indirectly regulates mmp-13 expression through the activation or repression of other factors , and further molecular and biological examination will be required .
ctgf and c - maf are co - localized in hypertrophic chondrocytes , and interestingly , c - maf - null and ctgf - null mice share common phenotypes , including expanded hypertrophic zones in the e16.5 embryo and reduced ossification of the tibia .
ctgf is expressed by proliferating and hypertrophic chondrocytes in the mouse growth plate , and it was identified as a c - maf - responsive gene in chondrocytes . 
 in vitro studies using reporter transfection assays have shown that c - maf and lc - maf activate the ctgf promoter , and chromatin immunoprecipitation assays demonstrated a direct interaction between the ctgf promoter and maf .
col2a1 was also upregulated by lc - maf via direct interactions with the intronic enhancer .
lc - maf is coexpressed with sox9 and col2a1 in the precartilaginous mesenchymal condensations during mouse embryogenesis , suggesting a role for lc - maf in early chondrocyte differentiation .
the same study identified an lc - maf binding site adjacent to the well - known sox9 binding site , although this site does not contain the typical mare consensus sequence .
the timing of in vivo expression of the sox proteins and lc - maf during chondrogenesis led to the hypothesis that lc - maf cooperates early with sox9 to activate target genes , including col2a1 , during mesenchymal condensation and that sox9/sox5/sox6 function at later steps of chondrocyte differentiation .
the enhancers of col27a1 contain a sox9-responsive element similar to that of col2a1 , and lc - maf and sox9 synergistically activate the expression of both col27a1 and col2a1 .
further evidence of the physiological significance of this activation comes from the co - localization of lc - maf and col27a1 in growth plate , with maximal expression of both genes occurring in the proliferating and pre - hypertrophic chondrocytes .
c - maf may be involved in chondrocyte apoptosis either in the normal bone formation process or in the progression of pathologic conditions .
the targets of c - maf in apoptosis pathways have been identified in nonchondrocyte cells and appear to be dependent on the specific cell context .
it has been shown that c - maf interacts with the transcription factor c - myb to downregulate bcl-2 expression and increase apoptosis in peripheral cd4 cells and that c - maf increases apoptosis by direct transactivation of caspase-6 in peripheral cd8 cells .
apoptosis through a p53-dependent pathway may also be relevant in both normal chondrocyte maturation and pathologic conditions .
the promoter of p53 contains mare consensus sequences , and overexpression of c - maf induces p53-dependent cell death in primary rat embryo fibroblast cells .
the p53-null embryonic mice have defects in the skeletal growth , abnormal chondrocyte maturation , and abnormal chondrocyte apoptosis , suggesting that p53 plays a role in normal chondrocyte development .
the target genes regulated by c - maf and lc - maf , as well as the phenotypes of c - maf - null mice , generate the general types of abnormalities commonly seen with factors that affect endochondral ossification or chondrocytic differentiation . a causative role for c
the maf proteins control multiple biological processes , and they are synthesized at varying levels and in distinct spatial - temporal patterns ; therefore , it is important to determine how they are regulated .
there must be regulation mechanisms that control the levels of maf members or splicing variants either on mrna and/or protein levels .
several upstream regulators of maf have been identified in the nonchondrocyte cells , and one can speculate that similar maf regulation exists in chondrocytes ( fig .
the fibroblast growth factor ( fgf ) pathway is a candidate for regulation of c - maf expression .
fgf signaling plays an important role in lens fiber differentiation , which is severely disrupted in c - maf - null mice .
deletion of three fgf receptors ( fgfr1 - 3 ) demonstrated that the expression of fgfr is required for normal lens development , and fgfr deletion is involved in reduced expression of c - maf and its target genes , crystallins , in the lens .
mapk pathway is activated upon interaction of fgfr and ligand , and the fgf / mapk cascade leads to the activation of erk1/2 kinases , which can phosphorylate mafa .
two conserved serine residues ( ser14 and ser65 ) located in the maf transactivation domains are phosphorylated by erk .
these phosphorylations affect the stability of maf and are required for maf - dependent transcriptional activation .
the transforming activity of mafa is controlled by glycogen synthase kinase3 ( gsk-3)dependent phosphorylations , at conserved serine / threonine residues that are also present on c - maf .
this phosphorylation resulted in the recruitment of coactivators to increase mafa - dependent transcription , and once released from the coactivators , the phosphorylation induced mafa ubiquitination and degradation .
similarly , glucocorticoids can regulate c - maf ubiquitination and proteosomal degradation to negatively regulate c - maf - dependent transcription , although the phosphorylation status of c - maf was not examined .
whether these pathways regulate c - maf in chondrocytes or during chondrogenic differentiation remains to be determined experimentally .
osteoarthritis ( oa ) is a degenerative joint disease characterized by erosion of the articular cartilage , hypertrophy of bone at the margins ( i.e. , osteophytes ) , subchondral sclerosis , and a range of biochemical and morphological alterations of the chondrocytes .
whereas endochondral ossification involves successive steps of chondrocyte maturation resulting in terminal hypertrophy and mineralization , normal articular chondrocytes are constrained from further differentiation , as evidenced by a lack of hypertrophic marker expression . however , during the progression of oa , the articular chondrocytes behave analogously to late hypertrophic and terminally differentiating growth plate chondrocytes with respect to their gene expression and cellular function .
increased expressions of hypertrophic chondrocyte markers and osteoblast markers occur in oa , such as type x collagen , osteopontin , osteocalcin , and alkaline phosphatase .
moreover , oa chondrocytes actively produce cartilage - degrading enzymes , including mmp-13 , which is normally expressed only in the late hypertrophic zone , and cell apoptosis is also frequently observed in oa cartilage .
thus , it has been suggested that oa may involve the functional loss of mechanisms that block articular chondrocyte maturation . in this context , understanding the mechanisms that control chondrocyte hypertrophy is important for normal skeletal development and growth , as well as cartilage degeneration during the pathogenesis of oa .
microarray analysis of a rat model of oa demonstrated an upregulation of mafb expression in degenerating cartilage .
it is noteworthy that the expression patterns of c - maf and mafb in rat embryo cartilage are virtually identical .
expression of c - maf was upregulated in oa cartilage compared to normal cartilage by real - time pcr and in situ hybridization . because it has been shown that cells in the chondrocyte clusters in oa have increased expression in both cartilage matrix proteins and matrix - degrading proteases , it is notable that c - maf mrna was detected in proliferating chondrocyte clusters in oa cartilage , whereas almost no c - maf - positive cells were found in normal cartilage .
the maf family proteins form a unique subclass of bzip transcription factors . since the identification of v - maf , many aspects of the physiological roles of maf proteins have been elucidated , and currently mafs are widely accepted as regulators of tissue - specific gene expression and cell differentiation .
studies of the roles of maf proteins in chondrocyte differentiation and cartilage are under way , as is the identification of genes directly regulated by c - maf .
the identification of c - maf binding partners and additional target genes during chondrogenesis and oa will provide insight into molecular functions of c - maf .
furthermore , studies to integrate upstream differentiation signal and modulation of c - maf activity at specific differentiation steps will be important to understanding chondrocyte terminal differentiation and oa pathogenesis .
 chondrocyte differentiation in the growth plate is an important process for the longitudinal growth of endochondral bones . 
 sox9 and runx2 are the most often - studied transcriptional regulators of the chondrocyte differentiation process , but the importance of additional factors is also becoming apparent . 
 mafs are a subfamily of the basic zip ( bzip ) transcription factor superfamily , which act as key regulators of tissue - specific gene expression and terminal differentiation in many tissues . 
 there is increasing evidence that c - maf and its splicing variant lc - maf play a role in chondrocyte differentiation in a temporal - spatial manner . 
 this review summarizes the functions of c - maf in chondrocyte differentiation and discusses the possible role of c - maf in osteoarthritis progression . 
Introduction
c-Maf Is a bZIP Transcription Factor
c-Maf in Chondrocytes
Interaction of Maf Proteins with Other Transcription Factors
c-Maf Target Genes and Pathways in Chondrocytes
Regulation of c-Maf
Osteoarthritis and Terminal Chondrocyte Differentiation
Concluding Remarks