interleukin 17 ( il-17 ) family of cytokines has been intensively investigated in recent years . as a proinflammatory cytokine , il-17 is involved in immune defense mechanisms acting through induction of secondary mediators of inflammation . however , its implication in various human inflammatory diseases such as rheumatoid arthritis and myositis was also demonstrated [ 13 ] . its receptor ( il-17r ) is ubiquitously expressed , in almost all cell types , allowing the widespread influence of il-17 on cellular processes , through subsequent activation of numerous signal transduction pathways , such as protein kinase a , jak / stat , nf-b , and mapks , including erk1/2 and p38 cascades [ 4 , 5 ] . known to be associated with the development of inflammatory diseases , il-17 has also been implicated in myopathies , such as polymyositis and dermatomyositis [ 7 , 8 ] . however , its role in muscle repair or regeneration has not been clarified yet . these cells have the capability to proliferate , migrate to sites of injury , and differentiate into adult muscle cells . during this process , the extracellular matrix ( ecm ) is dynamically and finely remodeled , while the imbalance in ecm reorganization can be involved in the development of muscle diseases . matrix metalloproteinases ( mmps ) are a family of enzymes which can selectively digest individual components of the ecm [ 10 , 11 ] . several mmps have been shown to be implicated in muscle regeneration , including matrix metalloproteinase type 2 ( mmp-2 ) and matrix metalloproteinase type 9 ( mmp-9 ) . in skeletal muscle , mmp-2 is constitutively expressed , whereas mmp-9 shows none to minimal basal expression [ 10 , 11 ] . in particular , mmp-9 expression and/or activity are tightly transcriptionally regulated and highly inducible in response to variety of agents , including proinflammatory cytokines . in addition , increased expression and activation of mmp-9 are associated with various myopathies and inflammation - induced changes in skeletal muscle . moreover , the upregulation of mmp-9 expression has been described in the muscles of mdx mice , an animal model of duchenne muscular dystrophy . even though high expression of both il-17 and mmp-9 has been reported in inflammatory myopathies , the mutual relation of these proteins in muscle is still not well understood . doxycycline ( doxy ) is an antibiotic of the tetracycline family of drugs and has been tested in numerous conditions associated with elevated mmp activity . it has recently been demonstrated that doxy can be beneficial for mdx mice mainly by reducing mmp-9 and tnf- expression . it was also demonstrated that doxy or anti - mmp-9 antibody improves soleus muscle regeneration and ameliorate development of excessive fibrosis . in addition , recent work showed that doxy treatment can regulate both local and systemic inflammation , suggesting its importance in inflammatory myopathies [ 17 , 18 ] . we have recently demonstrated the ability of il-17 to inhibit myogenesis in vitro , while mmp-9 participation as well as the regulatory role of doxy in this process has not been well defined so far . our findings indicate that il-17 increases mmp-9 expression , along with the inhibition of c2c12 myoblast differentiation . furthermore , results presented here show that doxy protects c2c12 myoblasts from the capacity of il-17 to inhibit myogenesis . myoblast c2c12 cell line was purchased from american type culture collection ( atcc , rockville , md ) . cells were cultured in growth medium ( gm ) consisting of dulbecco 's modified eagle 's medium ( dmem ) supplemented with 10% fetal bovine serum ( fbs ) from capricorn scientific ( ebsdorfergrund , germany ) and 100 units / ml penicillin and 0.1  mg / ml streptomycin ( capricorn scientific ) in a humidified environment at 37c and 5% co2 in air . myogenic differentiation was induced by culturing confluent cells in myogenic differentiation medium ( mdm ) consisting of gm supplemented with 2% horse serum instead of 10% fbs . recombinant mouse il-17 was provided by r&d systems ( minneapolis , mn , usa ) . doxycycline was from sigma - aldrich ( st . louis , mo , usa ) . mek1,2 inhibitor pd98059 was obtained from calbiochem ( darmstadt , germany ) and used at 25  m . 50.000  cells / well were seeded in 24-well plates and cultured overnight ; then , the cells were washed three times with pbs and 0.5  ml of serum - free culture medium was added . cells were subsequently cultured with or without il-17 as indicated for each experiment . during the induction of myogenic differentiation , cells were cultured for three days in mdm and additional 24  h to generate conditioned serum - free medium for mmp-9 determination . protein concentration in conditioned media was determined by bca protein assay macro kit ( serva , heidelberg , germany ) according to manufacturer 's instructions . aliquots of protein normalized conditioned media were subjected to 8% sds - page containing 0.1% gelatin under nonreducing conditions . gels were washed twice with 2.5% triton x-100 and rinsed one time with distilled h2o . the gels were then incubated for 24 hours in 100  mm tris - hcl , ph 8.5 , with 10  mm cacl2 . the activity of mmps was stopped by staining the gels with coomassie blue r250 in 50% methanol and 10% acetic acid for 20 minutes . zymography was developed by destaining gels in 20% methanol and 5% acetic acid until transparent bands were visualized . quantification of these areas was performed by densitometry analysis using nih - image j software . c2c12 cells were grown in 24-well plates until reaching 90% confluence and then subjected to myogenic differentiation by replacing gm with mdm . cell monolayers were washed twice with pbs and fixed with ice cold methanol for 2  min , then stained with crystal violet ( 0.1% ) for 15  min , and extensively washed with tap water . cultured cells were photographed using 10x objective lens and nih - image j software was used to measure myotube diameter . at least , 50 myotubes were used to determine the diameters and 3 short - axis measurements were taken along the length of a given myotube diameter and the average was calculated . antibodies against myosin heavy chain ( myhc ) , phospho - erk1/2 ( sc-7383 ) , and erk1 ( sc-94 ) were purchased from santa cruz biotechnology ( santa cruz , ca , usa ) . cells were lysed in lysis buffer ( 50  mm tris - hcl , ph 7.5 , 150  mm nacl , 1% np-40 ) containing 2  mm edta , 50  mm naf , and protease inhibitor cocktail ( serva electrophoresis gmbh , heidelberg , germany ) . equal amounts of protein from each sample were separated by sds - page and transferred to nitrocellulose membranes ( applichem , darmstadt , germany ) . membranes were blocked with 4% bsa in 0.5% tween-20 in tbs and then incubated with primary antibodies . membranes were then incubated with secondary antibodies conjugated with hrp ( sigma - aldrich ) . labeled proteins were visualized using enhanced chemiluminescence reagent system from applichem . protein bands were quantified by densitometry scanning , using nih - image j software and expressed relative to tubulin or corresponding total protein signals . two micrograms of total rna isolated from c2c12 cells was reverse transcribed using superscript ii ( invitrogen ) . pcrs were performed using one - step pcr ( invitrogen ) with the following settings : 94c for 5  min , 2630 cycles at 94c for 45  sec , 5254c for 30  sec , and 72c for 90  sec . the primer sets , annealing temperatures , number of cycles , and product size for each gene are given in supplementary table  1 ( in supplementary material available online at http://dx.doi.org/10.1155/2016/2939658 ) [ 22 , 23 ] . the reporter construct pmmp-9-luc containing 1300  bp of the 50-flanking region of the mouse mmp-9 gene has been previously described . zhenguo wu ( hong kong university of science & technology , hong kong , china ) . g133-luc and its derivatives were generated by inserting the xbai - bglii fragments from the corresponding chloramphenicol acetyltransferase constructs into the hindiii - bglii - digested pxp2 , respectively . a. corbi , cib , madrid , spain ) , which contains two copies of the c - fos sre ( nucleotides 357 to 275 , containing both an srf binding site and an adjacent ets motif ) upstream of a minimal tk promoter and the luciferase gene . pcmv--galactosidase expression vector was kindly provided by dr . c. bernabeu ( cib , spain ) and the constitutively active mutant of mek1 ( ca - mek1 ) was designed by substitution of the regulatory phosphorylation sites , ser218 and ser222 , with aspartic acid ( s218d / s222d mutant ) as described previously . c2c12 myoblasts seeded in 24-well plates ( 2  10  cells / well ) were transfected with different plasmids using turbofect transfection reagent according to the manufacturer 's protocol ( fermentas , st . after 24 hours of treatment , cells were lysed with 50  l of passive lysis reagent and firefly luciferase activity ( promega , madison , wi , usa ) was determined . -galactosidase activity ( tropix , bedford , ma , usa ) was measured as an internal control for transfection efficiency . statistical significance was evaluated using one - way anova followed by tukey 's test between individual groups . differences were considered significant at a value of p < 0.05 (  ) and p < 0.001 (  ) between control samples and treated samples . differences were considered significant at values p < 0.001 ( # # ) between il-17-treated samples and doxycycline plus il-17-treated samples . firstly , we examined whether il-17 can regulate the expression and production of mmp-9 , a major extracellular protease that can degrade several components of basement membrane and intramuscular connective tissue , in cultured c2c12 myoblasts . c2c12 cells were incubated in serum - free medium with increasing amounts of il-17 for 24 hours and the production of mmp-9 in culture supernatants was measured by gelatin zymography . the treatment with increasing amounts of il-17 significantly enhanced the activity of mmp-9 and increased mmp-9 mrna levels ( figure 1(a ) ) , while it did not produce detectable changes in mmp-2 activity ( supplementary figure  1(a ) ) . next , we evaluated the effect of il-17 on the transactivation of mmp-9 promoter in c2c12 cells . similar to the results obtained in zymography assay and rt - pcr , il-17 increased the transactivation of mmp-9-specific promoter in a dose - dependent manner ( figure 1(b ) ) . to determine whether il-17 affects mmp-9 expression during myogenic differentiation , c2c12 cells were induced to differentiate under il-17 treatment and mmp-9 expression was determined after three days . as presented in figure 2(a ) , both in gm and under differentiation induction , increased mmp-9 production and expression were noticed in response to il-17 treatment , as determined by both zymography and rt - pcr . moreover , during c2c12 differentiation induction , il-17 inhibited the expression of myogenesis markers myosin heavy chain ( myhc ) and muscle creatine kinase ( mck ) , determined by western blot and rt - pcr , respectively ( figure 2(b ) ) , suggesting that il-17 induces mmp-9 expression concomitantly with the inhibition of myogenic differentiation of c2c12 cells . the enhanced mmp-9 and reduced mck expression paralleled with the myotubes atrophy induced by il-17 , as shown in supplementary figure  1(b ) . modulation of mmp-9 is beneficial for the improvement of skeletal muscle regeneration in mdx mice [ 12 , 16 ] . it has been demonstrated that the mmp inhibitor doxy decreases the level of mmp-9 in biceps brachii and diaphragm of the mdx mice . next , we aimed to elucidate whether mmp-9 modulated by doxy may protect c2c12 cells from the inhibition of myogenesis induced by il-17 . the optimal concentration for doxy was set to 10  g / ml after dose - dependent experiment ( data not shown ) . doxy ( 10  g / ml ) improved myogenesis ( figure 3(a ) ) by producing more hypertrophic myotubes , determined by the average myotube diameter ( 2.45-fold compared with cells grown in mdm only ) . in addition , doxy protected myotubes formation in the presence of il-17 , as the cells had the hypertrophy index similar to that in control . interestingly , myotubes appeared to be shorter in the presence of il-17 than myotubes formed without il-17 treatment . the effect of doxy was confirmed by muscle - specific protein myhc expression . as shown in figure 3(b ) , myhc was highly expressed in the presence of doxy during myogenesis induction , while , in the presence of il-17 , c2c12 cells expressed myhc at similar levels as cells cultivated in mdm only . moreover , il-17-induced repression of the mck gene expression ( figure 3(c ) ) was completely conversed by doxy as well as the il-17-induced repression of myogenin promoter ( pg133-luc ) transactivity ( figure 3(d ) ) . beyond its capacity next , we determined whether doxy can transcriptionally regulate il-17-induced mmp-9 expression in c2c12 cells . as shown in figure 4(a ) , doxy ( 5 or 10  g / ml ) inhibited both basal mmp-9 expression levels and the capacity of il-17 to increase the secreted activity of mmp-9 . this inhibition seems to be due to transcriptional regulation , since doxy also reduced both promoter transactivation and mmp-9 mrna expression induced by il-17 ( figures 4(b ) and 4(c ) ) . moreover , similar doxy concentrations did not significantly modify mmp-2 secreted activity ( supplementary figure  1(c ) ) . subsequent experiments addressed the involvement of intracellular signaling pathways in il-17-induced mmp-9 expression in c2c12 cells . we have previously shown that il-17 activates erk1/2 in c2c12 cells , and , in order to clarify whether erk1/2 pathway mediates il-17-induced mmp-9 production and expression , c2c12 cells were treated with il-17 in the presence of mek1,2 inhibitor , pd98059 , and the mmp-9 protein and mrna expression were determined . as shown in figure 5(a ) , cotreatment of the cells with il-17 and mek1,2 inhibitor significantly blocked the increment of both mmp-9 activity and mrna expression induced by il-17 . furthermore , the il-17-increased mmp-9-specific promoter transactivation was dramatically inhibited by dominant - negative ( dn ) mek1 promoter , while the cotransfection with constitutively active mek1 , independently of il-17 , significantly transactivated the mmp-9 promoter ( figure 5(b ) ) . erk1/2 signaling appeared to be necessary and sufficient to mediate il-17-induced mmp-9 expression in c2c12 cells . the appropriate expression of both dominant - negative and constitutively active ( ca ) mutants of mek1 in transiently transfected c2c12 cells was confirmed by their reactivity to anti - ha antibody ( figure 5(c ) ) . it has also been shown that , independently of its capacity to inhibit mmp activity , doxy inhibits mapk activation . since erk1/2 signaling seems to be critical for il-17 's induction of mmp-9 , we determined whether doxy affects the activation of this pathway by il-17 . as shown in figure 5(d ) , the capacity of il-17 to induce erk1/2 phosphorylation was dramatically inhibited in the presence of doxy ( 5 or 10  g / ml ) . this result paralleled with the transactivation capacity of this signal , since doxy inhibited the capacity of il-17 to induce the transactivation of psre - luc reporter ( figure 5(e ) ) , thus indicating that doxy inhibits il-17-induced mmp-9 by downregulating erk1/2 signal transduction . coordination between inflammation and regeneration is believed to be crucial for the beneficial outcome of the repair process following muscle damage [ 28 , 29 ] . during this process , the extracellular matrix surrounding skeletal muscle cells has an important role in maintaining the structure of the muscle , acting as a scaffold for myofiber regeneration [ 10 , 30 ] . therefore , remodeling of ecm is important for muscle maintenance and repair in normal and pathological conditions . in addition to high levels of il-17 , elevated levels of mmp-9 have also been observed in skeletal muscle during inflammatory myopathies . however , regulation of mmp-9 expression by il-17 in muscle cells has not been elucidated yet . in the present work , our results demonstrated that il-17 increases the expression and production of mmp-9 in mouse myoblast cells c2c12 , while not significantly affecting the constitutively expressed mmp-2 ( figure 1 and supplementary figure ) . expression of mmp-2 and mmp-9 has been previously reported in myogenic cells of various species with a general consensus about the constitutive expression of mmp-2 by muscle cells and more controversial mmp-9 expression . our results obtained using c2c12 cells showed that il-17 increases mmp-9 expression at both transcriptional and protein level . since il-17 is primarily a proinflammatory cytokine , this result is in agreement with previous observations that mmp-9 expression is related to the inflammatory response , as well as that mmp-9 upregulation in muscle tissue appears to be a common finding in all inflammatory myopathies . in addition , it is consistent with the abundant evidence , indicating that mmp-9 gene expression is , to a large extent , tightly regulated at transcriptional level . furthermore , it is known that proinflammatory cytokines are able to activate muscle satellite cells , possibly by inducing mmp-9 expression . during myogenic differentiation , basal mmp-9 expression was increased and its expression was higher in the presence of il-17 . this effect was accompanied with the capacity of il-17 to inhibit the expression of myogenic differentiation marker mck , demonstrating that il-17 enhances mmp-9 expression concomitantly with the inhibition of myogenic differentiation of c2c12 cells . it could be speculated that chronic exposure to il-17 , by inducing mmp-9 expression , may affect the homeostasis of muscle cells , as well as their ecm remodeling , which could then contribute to the development of inflammatory muscle diseases . in pathological conditions , abnormal increment in mmp-9 expression and activity may produce excessive degradation of type iv collagen in the skeletal muscle basement membrane , which in cooperation with other mmps can lead to skeletal muscle tissue loss [ 10 , 33 ] . doxycycline is the most potent mmp inhibitor among the food & drug administration usa / health canada - approved tetracyclines , which inhibits mmp activity at plasma levels lower than those needed for its antimicrobial effect [ 34 , 35 ] . in addition , doxy , beyond its role in proteolysis , possesses biological roles in inflammation , angiogenesis , apoptosis , metal chelation , ionophoresis , and bone metabolism . our results demonstrated that doxy may rescue in vitro myoblast differentiation by reverting il-17 inhibition of myogenesis and mmp-9 induction . moreover , during the induction of myoblastic differentiation , doxy appeared to increase myotube mass , indicated by the hypertrophic index and myhc expression . interestingly , although mmp-9 inhibition was shown to improve soleus muscle regeneration in vivo , it has been reported by zimowska et al . that doxy may delay c2c12 differentiation in vitro . in the aforementioned study , authors used doxy at concentration of 60  m which is approximately three times higher than the highest concentration utilized in this study ( 10  g / ml or 22  m ) . we observed that 5  g / ml ( 11  m ) is enough to induce myotube hypertrophy . this may indicate that the doxy concentration is important to achieve the desired effects , and higher concentration may impair myogenesis by excessive inhibition of mmp-9 , which is also necessary for normal myogenic differentiation where mmp-9 is mainly secreted during prefusion stages . however , during inflammatory process , increased mmp-9 secretion may disturb the proteolytic homeostasis , thus impairing muscle cell differentiation , as observed here for il-17 . as mentioned above , doxycycline may also affect different biological processes which can be explained by its capacity to influence the activation of intracellular signals such as erk1/2 signaling [ 27 , 37 ] . in this line , il-17 was shown to trigger numerous signal transduction pathways , including erk1/2 and p38 mapks . in accordance with our previous work , we confirmed here that il-17 is able to enhance phosphorylation of erk1/2 in c2c12 cells , and we also showed that this pathway was essential for the induction of mmp-9 expression by il-17 . furthermore , we demonstrated that mek1,2-erk1/2 inhibition by using the chemical inhibitor pd98059 reverted the suppressive effect of il-17 on c2c12 myogenic differentiation . moreover , pd98059 was shown to enhance c2 myoblast cell differentiation in vitro , thus suggesting that doxy may rescue c2c12 myogenic differentiation by downregulating il-17 activation of erk1/2 signaling which is necessary for the increased mmp-9 expression . interestingly , il-17 did not affect the capacity of c2c12 cells to secrete mmp-2 , and similar result was obtained by treating cells with doxy ( supplementary figure ) , indicating that mmp-2 is not a target gene neither for il-17 nor for doxy . it has been demonstrated that during c2c12 differentiation mmp-2 expression is mainly unaltered , since mmp-2 is expressed in these cells by a constitutive mechanism [ 39 , 40 ] . although , in some inflammatory myopathies , such as polymyositis , dermatomyositis , and inclusion body myositis , both mmp-2 and mmp-9 are upregulated , their balance depends on the type of disease and local presence of various factors . in this regard , upregulated mmp-9 in muscle tissue appears to be a common finding in all inflammatory myopathies , while mmp-2 seems to be affected to a lesser extent . results presented here , together with our previously published results , which demonstrated that il-17 does not affect mmp-2 expression , while it inhibits the expression of upa in c2c12 cells , further confirm the complex role that il-17 has on proteolytic enzymes expression and activity in myoblasts . therefore , il-17 may affect c2c12 myogenic differentiation by increasing mmp-9 expression , while doxy can protect and preserve mmp-9/mmp-2 balance during myogenesis via erk1/2 by reducing mmp-9 expression . of note , it would be of interest to explore the relative ratio between mmps and tissue inhibitors of mmps ( tipms ) , as there is no available data about this mode of regulation during muscle inflammation and regeneration involving il-17 , although there are some reports about their mutual regulation during muscle differentiation . finally , in duchenne muscular dystrophy ( dmd ) , primary deficiency of dystrophin leads to several secondary pathological changes including ecm breakdown , inflammation , and fibrosis , all of which require mmp activity . besides revealing that mmp-9 inhibition augments the proliferation of satellite cells and changes the immune cell milieu as well as expression of significant ligands , receptors , and signaling pathways , this group showed that inhibition of mmp-9 dramatically improves the engraftment of transplanted myoblasts in skeletal muscle of mdx mice ( a mouse model of dmd ) . together with our results , this finding adds value to the importance of mmp-9 in muscle regeneration and suggests this enzyme as a possible target in muscle regeneration and repair . however , the effect of il-17 in the regulation of mmp-9 production in myoblast cells and its association with physiological events and/or pathological conditions as well as doxycycline inhibition of il-17-induced mmp-9 expression should be additionally confirmed in further in vivo studies , as this will help determine its role in muscle disease and regeneration . our data provide evidence that doxycycline inhibits the capacity of il-17 to induce mmp-9 expression in myoblast cells by regulating the activation of erk1/2 parallel with its ability to protect c2c12 myoblast cells from the inhibitory effect that il-17 has on myogenic differentiation . the regulation of il-17induced mmp-9 expression by doxycycline may be useful to control the proteolytic status during muscle regeneration and muscle inflammatory diseases .
interleukin 17 ( il-17 ) is a cytokine with pleiotropic effects associated with several inflammatory diseases . 
 although elevated levels of il-17 have been described in inflammatory myopathies , its role in muscle remodeling and regeneration is still unknown . 
 excessive extracellular matrix degradation in skeletal muscle is an important pathological consequence of many diseases involving muscle wasting . in this study , 
 the role of il-17 on the expression of matrix metalloproteinase- ( mmp- ) 9 in myoblast cells was investigated . 
 the expression of mmp-9 after il-17 treatment was analyzed in mouse myoblasts c2c12 cell line . 
 the increase in mmp-9 production by il-17 was concomitant with its capacity to inhibit myogenic differentiation of c2c12 cells . 
 doxycycline ( doxy ) treatment protected the myogenic capacity of myoblasts from il-17 inhibition and , moreover , increased myotubes hypertrophy . 
 doxy blocked the capacity of il-17 to stimulate mmp-9 production by regulating il-17-induced erk1/2 mapk activation . 
 our results imply that mmp-9 mediates il-17 's capacity to inhibit myoblast differentiation during inflammatory diseases and indicate that doxy can modulate myoblast response to inflammatory induction by il-17 .