BACKGROUND
adipose tissue is no longer considered as a mere energy reservoir but it plays also an endocrine role, releasing a panoply of secreted molecules, i.e.
adipokines such as leptin, adiponectin, plasminogen activator inhibitor  <dig> , vaspin and tumor necrosis factor α   <cit> .
furthermore, adipose tissue is a source of stem cells, representing a promising tool for pharmacological studies and clinical applications  <cit> .
a balanced development of adipose tissue is of crucial importance to ensure some of the most important physiological functions, including reproduction, haemostasis, angiogenesis, blood pressure and immune function  <cit> .
alterations of fat cell number and size are present in lipodystrophy and obesity that are associated to type  <dig> diabetes  <cit> .
another condition altering fat cell formation is osteoporosis, where an imbalance between adipocytes and osteoblasts in bone marrow is observed.
aging, menopause, glucocorticoid treatment or alcohol abuse can lead to an increase in bone marrow adiposity  <cit> .
to date, several issues are still pending, for instance whether infiltration of fat in bone marrow causes low bone mass or is due to bone loss  <cit> .
since adipocytes and osteoblasts share the same mesenchymal precursor, the study of the adipocyte/osteoblast balance represents a worthy challenge to treat adipose tissue and bone disorders.
it is well described that secreted leptin and adiponectin can affect bone formation both directly on osteoblastogenesis and indirectly by acting on osteoclastogenesis  <cit> .
several molecules secreted by osteoblasts such as wnt and bone morphogenetic protein favor osteogenesis at the expense of adipogenesis  <cit> , thus pointing out a crosstalk between both lineages.
during the last two decades, a large number of molecular regulators of adipogenesis and osteogenesis have been described.
among them, peroxisome proliferator-activated receptor γ  and ccaat/enhancer-binding proteins  are well recognized factors that play major roles in adipogenesis  <cit> , whereas runt-related transcription factor  <dig> , distal-less homeobox  <dig> , muscle segment homeobox  <dig>  and osterix represent master regulators of osteogenesis  <cit> .
in the present work, we aimed at identifying molecules secreted at early step of differentiation of human mesenchymal stem cells towards adipocytes and osteoblasts.
to address this point, we used a cellular model recently established in our laboratory, termed hmads cells .
hmads cells, isolated from the adipose tissue of young donors, present extensive capacities of self-renewal, clonogenicity and multipotency, as they fully differentiate into adipocytes, osteoblasts, myoblasts and chondrocytes while exhibiting a normal karyotype  <cit> .
recently, proteomic approaches have been applied to study rodent and human adipose tissue secretome using cellular models that focused mainly on late events of adipogenesis  <cit> .
moreover, with respect to early events of osteogenesis, a characterization of secreted molecules from osteoblasts has not been so far reported.
herein we have identified  <dig> proteins by a proteomic approach and confirmed these findings by western-blot for  <dig> candidates.
these proteins and their involved pathways, in particular the plasminogen system, could play an important role in regulating the adipocyte/osteoblast balance.
RESULTS
characterization of the secretome of hmads cells committed towards adipocytes and osteoblasts
hmads cells differentiate into adipocytes and osteoblasts, as shown in figure 1a.
gene expression of representative markers of adipocyte  and osteoblast differentiation  are reported and in agreement with previously published data  <cit> .
the terminal differentiation of hmads cells into adipocytes and osteoblasts is also illustrated by the typical cellular morphology as shown in figure 1b.
in order to analyze the secretome at early step of adipogenesis and osteogenesis of hmads cells, secretion media at day  <dig> of differentiation into adipocytes and osteoblasts were compared with those of cells at day  <dig>  cells were washed  <dig> times with pbs and incubated for  <dig> h with serum-free culture medium, containing  <dig>  μg/ml of transferrin without differentiation inducers.
at the end of this incubation period, media were harvested, filtered and concentrated.
very low cell death or cell lysis occurred during the incubation period, as checked by microscopic analysis and lactate dehydrogenase activity measurements .
four μg of each protein sample were loaded onto one-dimensional sds-page and stained with bio-safe coomassie stain.
a representative gel is reported in figure 1c.
the excised bands from the sds-page were cut, trypsin digested and analyzed by mass spectrometry.
the list of the identified proteins is reported in table  <dig>  further details about the peptides identified for each protein are available in table 1s .
y: indicates the presence of the candidate in the sample; sp: signal peptide; ex: extracellular; in: intracellular; mw: molecular weight; adipo:day  <dig> adipocytes; osteo: day  <dig> osteoblasts; loc: localisation; ref: reference.
* indicates only putatively
seventy three proteins were classified according to their function and localization, determined by the meta search engine bioinformatic harvester.
as reported in figure 2a and table  <dig>  a large amount of identified proteins  corresponded to "classical" secreted proteins characterized by a signal peptide.
among them, we found several components of the extracellular matrix , proteases, serine protease inhibitors  and plasma protease c <dig> inhibitor) and other proteins involved in collagen catabolism, protein folding/turnover and growth regulation.
in differentiating osteoblasts at day  <dig>  periostin, described previously to be associated with osteoblast differentiation  <cit>  was identified, whereas its presence was not detected in secretion media of differentiating adipocytes.
a high percentage of proteins , devoid of signal peptide, was also detected into the secretion medium.
these proteins were labelled as extracellular since they have been previously described in the extracellular compartment.
some of these proteins were metabolic enzymes such as α-enolase  and pyruvate kinase, cytoskeletal components or heat shock/chaperone proteins.
similar findings from literature have been reported  strengthening actually their presence in the extracellular medium of various cellular models as well as in the murine/human bloodstream.
the remaining proteins  did not exhibit a signal peptide  and, to the best of our knowledge, have not been so far described extracellularly and did not represent artefacts as cell lysis was very low .
these proteins are only putatively secreted and indicated by an asterisk in table  <dig>  as illustrated by the venn diagram in figure 2b, the secretion of  <dig> proteins resulted to be similar under the three culture conditions, while  <dig> proteins were specifically secreted under adipogenic condition compared to  <dig> proteins under osteogenic condition.
finally, hmads cell secretome was clustered in  <dig> main groups, as indicated in table  <dig> and figure  <dig>  these clusters include: proteases, protease inhibitors, extracellular matrix  components, anti-inflammatory/anti-oxidant proteins, metabolic enzymes, cytoskeletal components, heat shock/protein folding proteins and other proteins.
these clusters illustrated the diversity of hmads cell secretome, thus suggesting that some of these proteins could be involved in the regulation of the adipocyte/osteoblast balance.
validation of protein expression of selected candidates
mass spectrometry identification data were validated for  <dig> candidates, selecting them from different clusters, i.e.
<dig> protease,  <dig> protease inhibitors,  <dig> ecm components,  <dig> anti-inflammatory/anti-oxidant protein,  <dig> metabolic enzyme and  <dig> heat shock protein.
western blots or zymograms were employed to supplement the identification by mass spectrometry and to determine the expression profile among three different sets of conditions .
for all the candidates, the results of immunoblotting/zymogram and a semi-quantification of the bands are reported in figure  <dig>
matrix metallopeptidase  <dig> , secreted protein, acidic, cysteine-rich  and eno <dig> were secreted by hmads cells under all conditions, confirming the pattern of detection obtained by sds-page-mass spectrometry analysis .
however, no significant difference was observed under the three conditions for these candidates.
zymographic analysis of the secretion media revealed a prominent band of lysis at  <dig> kda corresponding to the reported active form of mmp <dig> .
addition of edta, an inhibitor of gelatinase activity, led to the absence of lytic bands , thus confirming the specificity of mmp activity.
pigment epithelium derived factor  was highly expressed in day  <dig> adipocytes compared to day  <dig> and day  <dig> osteoblasts  in agreement with mass spectrometry identification.
for  <dig> kda glucose-regulated protein precursor , transforming growth factor-beta-induced protein , pentraxin  <dig>  and pai- <dig>  we confirmed the pattern of detection reported in table  <dig>  with significant differences of expression between day  <dig>  day  <dig> adipocytes and day  <dig> osteoblasts, as shown by western blot and histogram semi-quantifications .
analysis of the plasminogen system in hmads cells differentiating towards adipocytes and osteoblasts
given the pattern of expression at the early step of adipogenesis and osteogenesis of hmads cells, pai- <dig> appeared as one of the most promising candidates for investigating further the adipocyte/osteoblast balance.
pai- <dig> mrna levels were similar to those of protein levels .
since pai- <dig> is a key regulator of the conversion of plasminogen to plasmin, negatively acting on two plasminogen activators, namely upa  and tpa   <cit> , we decided to evaluate upa and tpa levels in hmads cell secretome.
elisa experiments were performed on secretion media from hmads cells at day  <dig> and day  <dig> under adipogenic and osteogenic differentiating conditions after  <dig> h of incubation.
as reported in figure 5b, consistent with the immunoblotting data, pai- <dig> levels strongly decreased in differentiating adipocytes as compared to day  <dig>  by contrast, pai- <dig> was secreted by day  <dig> osteoblasts, at albeit the same levels when compared to day  <dig>  concerning tpa measurement, we did not detect tpa in day  <dig> differentiating adipocytes, while a significant increase of tpa levels was observed in day  <dig> osteoblasts as compared to day  <dig> .
by contrast, upa remained undetectable in the secretion media of hmads cells under the three conditions analyzed.
enzymatic activities of upa and tpa in  <dig> h serum-free conditioned media of hmads cells at d <dig> and day  <dig> of differentiating adipocytes and osteoblasts were then evaluated by casein/plasminogen zymographic analysis.
as reported in figure 5d, tpa was mostly secreted by day  <dig> osteoblasts, as observed by the lytic band at  <dig> kda, while upa was undetectable , thus supporting elisa results.
the higher band at around  <dig> kda may correspond to complexes between plasminogen activators and pai- <dig> as classically reported  <cit> .
incubation of the gel with amiloride, which selectively abrogates upa-dependent enzyme activities, did not affect tpa-dependent lytic bands .
furthermore, we demonstrated that hmads cells expressed upa receptor  mrna as reported by semi-quantitative rt-pcr analysis .
as these data actually suggest the involvement of plasminogen activation in the regulation of the fine balance between adipogenesis and osteogenesis, this hypothesis was next examined in a pathophysiological situation where such an imbalance occurs.
pai-i expression in ovariectomized mice
disequilibrium of the adipocyte/osteoblast balance is associated with the development of osteoporosis after ovariectomy.
it is well established that in the bone of ovariectomized  mice, adipocytes develop at expense of bone formation  <cit> .
in order to evaluate whether osteoporosis affects pai- <dig> in bone, we analyzed the expression of pai- <dig> mrna in the humerus of ovx compared to sham mice.
ten weeks after surgery, as previously described  <cit>  mice increased fat mass as shown by leptin levels .
as shown in figure 6b, the levels of pai- <dig> mrna decreased significantly in the humerus of ovx compared to sham mice, thus pointing out at pai- <dig> modulation in the adipocyte/osteoblast balance as a potential marker of osteoporosis.
discussion
characterization of hmads cell secretome
the secretome of murine and human adipocytes has been characterized  <cit> .
however, these studies primarily focused on the late events of adipogenesis.
concerning osteoblast secretome, a single detailed characterization has been performed so far by xiao and collaborators, who analyzed the extracellular matrix vesicle proteome of mineralizing osteoblasts and identified proteins at late steps of osteogenesis of mc3t3-e <dig> cells  <cit> .
herein, proteomic analysis has been used for the first time to compare the secretome of hmads cells differentiating into adipocytes and osteoblasts and to gain insights into the adipocyte/osteoblast balance.
in order to delineate the initial secretory events of adipogenesis and osteogenesis, we performed a  <dig> h short term secretion which likely limits the number of secreted proteins and cell lysis.
a total of  <dig> proteins was identified and classified into  <dig> clusters.
the largest cluster corresponds to extracellular matrix proteins, thus confirming the secretome characteristic of our fraction.
in addition, more than 50% of hmads cell secretome is represented by proteins with signal peptide.
cytoskeletal proteins and heat shock and folding proteins are also largely represented with  <dig> and  <dig> proteins, respectively.
among the heat shock/folding proteins, two chaperones, e.g.
hsp <dig> and hsp <dig> were found.
these findings are reinforced by recent observations describing the presence of hsp <dig> and hsp <dig> in the bloodstream.
hsp <dig> circulating levels in diabetic patients have been found associated to cardiovascular diseases  <cit> , while elevated hsp <dig> circulating levels are related to systemic inflammatory reaction in patients with heart failure following acute myocardial infarction  <cit> .
in the metabolic enzyme cluster,  <dig> enzymes were also found, including glyceraldehyde dehydrogenase  and phosphoglycerate kinase .
according to literature, extracellular gapdh, also found in rat serum, inhibits spreading of cos- <dig> cells  <cit> , while pgk is secreted by tumor cells and its activity is regulated by hypoxia  <cit> .
these enzymes might reflect increased metabolic requirements, but they could also exert independent and still undiscovered functions.
anti-oxidant proteins, like thioredoxin reductase  <dig> , represent another cluster of proteins secreted by hmads cells.
txnrd <dig>  that does not exhibit a signal peptide, has been described in rat as a circulating protein  <cit> .
eleven percent of secreted proteins from hmads cells is however represented by intracellular proteins, presence of which has not been reported up to now outside the cells.
a modest contamination by uncontrolled release of intracellular proteins cannot be excluded despite the fact that no cell lysis was detected.
although cell death can take place in culture and is unavoidably accompanied by a subsequent release of cytosolic components, it should be pointed out that cell viability was not affected .
among non-artifactual explanations, it is worth pointing out that this phenomenon could implicate exosome secretion, as most of the identified proteins were reported to be present in exosomes.
exosomes are membrane vesicles that originate from the cell membrane and are released extracellularly  <cit> .
their occurrence has been also described in the bloodstream and in different tissues in vivo  <cit> , suggesting their participation in physiological and/or pathological processes.
to date, the exact function of exosomes in vivo is not completely understood.
a large number of cell types, including reticulocytes, immune cells, platelets, epithelial cells, dendritic cells and others  appears to secrete exosomes either as a mechanism to extrude some proteins and/or to communicate between cells and/or to play immune functions.
typically, exosomes contain chaperones, cytoskeletal proteins , elongation factors and several enzymes , as described in literature through proteomic analysis of the secretion media from different sources as well as from analysis by flow cytometry and western blot  <cit> .
therefore intracellular proteins recovered outside hmads cells may be a mere reflection of events taking place within the cytosol at the time of exosome formation.
furthermore,  <dig>  out of  <dig> candidates, known to be intracellular, have a score higher than a fixed threshold when analyzed using software predicting a non-classical protein secretion  <cit> , it is likely that these proteins are secreted by non-traditional mechanisms and did not arise from cell lysis.
clearly, these proteins can only be considered as putatively secreted and their actual secretion should be assessed.
selected candidates of hmads cell secretome
mass spectrometry data have been validated for  <dig> selected candidates.
mmp <dig>  sparc and eno <dig> displayed no significant differences in terms of quantification by immunoblotting or zymogram when comparing the three culture conditions.
mmp <dig>  a zinc-dependent endopeptidase, involved in the degradation of the ecm, is actively secreted by adipocytes  <cit>  and osteoblasts  <cit> .
the presence of mmp <dig> in hmads cells reinforces the concept that ecm remodelling represents a crucial event during the early steps of differentiation.
as observed in this study, sparc has been already described as a secreted molecule in both adipocytes  <cit>  and osteoblasts  <cit> .
sparc is reported to be up-regulated in the adipose tissue of different models of murine obesity  <cit>  and its circulating levels correlate with body mass index in humans  <cit> .
a potential interest in further studying sparc in the adipocyte/osteoblast balance relies upon the fact that bone marrow stromal cells from sparc-null mice tend to form more adipocytes than cells from wild type mice  <cit> .
eno <dig> is mainly described for its role as an intracellular glycolytic enzyme, however its presence extracellularly has been already reported in the secretion medium of 3t3-l <dig> adipocytes  <cit> , as well as in other cellular models  <cit> .
eno <dig> is a multifunctional protein and a putative plasminogen receptor, since it has been reported as a cell surface protein  <cit> .
therefore, a potential role of eno <dig> in the plasminogen cascade cannot be excluded.
concerning the five other candidates , relevant differences of expression between day  <dig> and day  <dig> of adipogenesis and osteogenesis have been found.
pedf has been reported to be preferentially secreted by 3t3-l <dig> preadipocytes as compared to fully differentiated adipocytes  <cit> .
however, zvonic et al.
<cit>  have described an increase in pedf expression during adipogenesis of primary cultures of human adipose-derived stem cells, in agreement with our observations in hmads cells when comparing hmads cells at day  <dig> and after complete adipocyte differentiation .
pedf could represent a valuable candidate for the adipocyte/osteoblast balance given its expression also in differentiating hmads osteoblasts.
a putative role for pedf as mediator of angiogenesis and in matrix remodelling of the bone has been recently postulated  <cit> .
several molecular chaperones have been found in the secretome of hmads cells, including calumenin, calreticulin, protein disulfide isomerase  <dig>  heat shock proteins and grp <dig>  as grp <dig> controls intracellular protein transport  <cit> , its up-regulation in hmads differentiating adipocytes and osteoblasts as compared to day  <dig> could reflect an increased demand in protein folding or a protection against endoplasmic reticulum stress.
bigh <dig> is reported to be down-regulated during the differentiation of murine osteoblasts and to play negative effects on the early stages of osteogenesis  <cit> .
in hmads cells, bigh <dig> protein levels increase in differentiating osteoblasts as compared to day  <dig>  however a decrease during osteoblast terminal differentiation was observed .
interestingly, bigh <dig> is also released by differentiating adipocytes at day  <dig>  so far, no report has described the secretion of bigh <dig> by adipocytes or adipose tissue, whereas bigh <dig> is involved in distinct cellular functions, such as cell growth, tumorigenesis, wound healing, apoptosis and migration  <cit> .
thus a role for bigh <dig> in adipogenesis remains to be established.
ptx <dig> is a member of the pentraxin family that we described to be secreted by adipocytes and to respond to inflammatory stimuli such as tnfα  <cit> .
in differentiating hmads cells, ptx <dig> appears up-regulated during both adipogenesis and osteogenesis.
the presence of ptx <dig> in osteoblasts has never been reported; we speculate that, under conditions of stress or inflammation, ptx <dig> could represent a potential target of the adipocyte/osteoblast balance that might deserve further studies.
finally, pai- <dig> represents another member of the serpin family which is secreted by hmads cells.
pai- <dig> is a pleiotropic molecule, exerting functional roles in wound healing, atherosclerosis, tumor angiogenesis, rheumatoid arthritis, fibrosis etc., besides its main role as regulator of fibrinolysis  <cit> .
in addition, pai- <dig> plays a role in metabolic disorders such as obesity and insulin resistance, representing a marker of metabolic syndrome  <cit> .
a role for pai- <dig> in bone remodeling has been postulated, since mice lacking pai- <dig> are protected from trabecular bone loss after ovariectomy, suggesting a site-specific role for pai- <dig> in bone turnover  <cit> .
altogether, these data indicate that pai- <dig> should represent a reliable candidate for the study of the adipocyte/osteoblast balance.
in hmads cell secretome, pai- <dig> signal disappears at day  <dig> of adipogenesis while remaining present in differentiating osteoblasts.
despite some demonstration that pai- <dig> is produced by adipocytes during adipogenesis  <cit> , it was recently shown that adipogenesis, as such, may not induce pai-1; rather it enhances the potential of adipocytes to respond to pai- <dig> inducers  <cit> .
this may explain why pai- <dig> synthesis dropped after differentiation of human adipocytes cultured in the absence of serum, known to contain several pai- <dig> inducers  <cit> .
moreover overexpression of pai- <dig> by adenovirus-mediated gene transfer inhibited adipocyte differentiation  <cit> .
conversely, preadipocytes from pai-1-/- mice showed greater differentiation than those issued from wild type mice  <cit> .
this suggests that not only pai- <dig> is dispensable for adipocyte differentiation but could even be deleterious as it has been recently suggested that bone marrow-derived pai- <dig> had an effect on the development of obesity through its effect in inflammation  <cit> .
tpa is also preferentially secreted by hmads differentiating osteoblasts, while no detectable levels of upa have been detected, despite the presence of upar mrna.
interestingly, pai- <dig> and tpa serum levels have been reported as putative non-invasive diagnostic biomarkers of idiopathic osteonecrosis of the femoral head  <cit> .
as for circulating pai- <dig>  a correlation with dysbaric osteonecrosis has been also described  <cit> .
these data strongly suggest that the plasminogen/plasmin system could represent a potential target for further investigation of the adipocyte/osteoblast balance.
even if preliminary, our data concerning a decrease of pai- <dig> mrna levels in bone of ovx as compared to sham mice clearly indicate that pai- <dig> expression is modulated under conditions altering the adipocyte/osteoblast balance, such as osteoporosis.
CONCLUSIONS
in conclusion, we have characterized the secretome of hmads cells during the early events of adipogenesis and osteogenesis using a proteomic approach.
hmads cell secretome is represented by bona fide secreted proteins, proteins secreted through non-classical pathways and intracellular proteins, the presence of which in the extracellular medium being likely due to exosome secretion.
eight clusters have been identified, including proteases, protease inhibitors, ecm components, anti-inflammatory-antioxidant proteins, metabolic enzymes, cytoskeletal components, heat-shock/protein folding proteins and other proteins.
among the  <dig> selected proteins, pai- <dig> constitutes the most promising candidate for further studies owing to the role of the plasminogen/plasmin system in adipogenesis and bone remodeling.

 adipose tissue is no longer considered as a mere energy reservoir but it plays also an endocrine role, releasing a panoply of secreted molecules, i.e. adipokines such as leptin, adiponectin, plasminogen activator inhibitor, vaspin and tumor necrosis factor α. furthermore, adipose tissue is a source of stem cells, representing a promising tool for pharmacological studies and clinical applications. a balanced development of adipose tissue is of crucial importance to ensure some of the most important physiological functions, including reproduction, haemostasis, angiogenesis, blood pressure and immune function. alterations of fat cell number and size are present in lipodystrophy and obesity that are associated to type diabetes. another condition altering fat cell formation is osteoporosis, where an imbalance between adipocytes and osteoblasts in bone marrow is observed. aging, menopause, glucocorticoid treatment or alcohol abuse can lead to an increase in bone marrow adiposity. to date, several issues are still pending, for instance whether infiltration of fat in bone marrow causes low bone mass or is due to bone loss. since adipocytes and osteoblasts share the same mesenchymal precursor, the study of the adipocyte/osteoblast balance represents a worthy challenge to treat adipose tissue and bone disorders. it is well described that secreted leptin and adiponectin can affect bone formation both directly on osteoblastogenesis and indirectly by acting on osteoclastogenesis. several molecules secreted by osteoblasts such as wnt and bone morphogenetic protein favor osteogenesis at the expense of adipogenesis, thus pointing out a crosstalk between both lineages. during the last two decades, a large number of molecular regulators of adipogenesis and osteogenesis have been described. among them, peroxisome proliferator-activated receptor γ and ccaat/enhancer-binding proteins are well recognized factors that play major roles in adipogenesis, whereas runt-related transcription factor, distal-less homeobox, muscle segment homeobox and osterix represent master regulators of osteogenesis. in the present work, we aimed at identifying molecules secreted at early step of differentiation of human mesenchymal stem cells towards adipocytes and osteoblasts. to address this point, we used a cellular model recently established in our laboratory, termed hmads cells. hmads cells, isolated from the adipose tissue of young donors, present extensive capacities of self-renewal, clonogenicity and multipotency, as they fully differentiate into adipocytes, osteoblasts, myoblasts and chondrocytes while exhibiting a normal karyotype. recently, proteomic approaches have been applied to study rodent and human adipose tissue secretome using cellular models that focused mainly on late events of adipogenesis. moreover, with respect to early events of osteogenesis, a characterization of secreted molecules from osteoblasts has not been so far reported. herein we have identified proteins by a proteomic approach and confirmed these findings by western-blot for candidates. these proteins and their involved pathways, in particular the plasminogen system, could play an important role in regulating the adipocyte/osteoblast balance.  characterization of the secretome of hmads cells committed towards adipocytes and osteoblasts hmads cells differentiate into adipocytes and osteoblasts, as shown in figure 1a. gene expression of representative markers of adipocyte and osteoblast differentiation are reported and in agreement with previously published data. the terminal differentiation of hmads cells into adipocytes and osteoblasts is also illustrated by the typical cellular morphology as shown in figure 1b. in order to analyze the secretome at early step of adipogenesis and osteogenesis of hmads cells, secretion media at day of differentiation into adipocytes and osteoblasts were compared with those of cells at day cells were washed times with pbs and incubated for h with serum-free culture medium, containing μg/ml of transferrin without differentiation inducers. at the end of this incubation period, media were harvested, filtered and concentrated. very low cell death or cell lysis occurred during the incubation period, as checked by microscopic analysis and lactate dehydrogenase activity measurements. four μg of each protein sample were loaded onto one-dimensional sds-page and stained with bio-safe coomassie stain. a representative gel is reported in figure 1c. the excised bands from the sds-page were cut, trypsin digested and analyzed by mass spectrometry. the list of the identified proteins is reported in table further details about the peptides identified for each protein are available in table 1s. y: indicates the presence of the candidate in the sample; sp: signal peptide; ex: extracellular; in: intracellular; mw: molecular weight; adipo:day adipocytes; osteo: day osteoblasts; loc: localisation; ref: reference. * indicates only putatively seventy three proteins were classified according to their function and localization, determined by the meta search engine bioinformatic harvester. as reported in figure 2a and table a large amount of identified proteins corresponded to "classical" secreted proteins characterized by a signal peptide. among them, we found several components of the extracellular matrix, proteases, serine protease inhibitors and plasma protease c inhibitor) and other proteins involved in collagen catabolism, protein folding/turnover and growth regulation. in differentiating osteoblasts at day periostin, described previously to be associated with osteoblast differentiation was identified, whereas its presence was not detected in secretion media of differentiating adipocytes. a high percentage of proteins, devoid of signal peptide, was also detected into the secretion medium. these proteins were labelled as extracellular since they have been previously described in the extracellular compartment. some of these proteins were metabolic enzymes such as α-enolase and pyruvate kinase, cytoskeletal components or heat shock/chaperone proteins. similar findings from literature have been reported strengthening actually their presence in the extracellular medium of various cellular models as well as in the murine/human bloodstream. the remaining proteins did not exhibit a signal peptide and, to the best of our knowledge, have not been so far described extracellularly and did not represent artefacts as cell lysis was very low. these proteins are only putatively secreted and indicated by an asterisk in table as illustrated by the venn diagram in figure 2b, the secretion of proteins resulted to be similar under the three culture conditions, while proteins were specifically secreted under adipogenic condition compared to proteins under osteogenic condition. finally, hmads cell secretome was clustered in main groups, as indicated in table and figure these clusters include: proteases, protease inhibitors, extracellular matrix components, anti-inflammatory/anti-oxidant proteins, metabolic enzymes, cytoskeletal components, heat shock/protein folding proteins and other proteins. these clusters illustrated the diversity of hmads cell secretome, thus suggesting that some of these proteins could be involved in the regulation of the adipocyte/osteoblast balance. validation of protein expression of selected candidates mass spectrometry identification data were validated for candidates, selecting them from different clusters, i.e. protease, protease inhibitors, ecm components, anti-inflammatory/anti-oxidant protein, metabolic enzyme and heat shock protein. western blots or zymograms were employed to supplement the identification by mass spectrometry and to determine the expression profile among three different sets of conditions. for all the candidates, the results of immunoblotting/zymogram and a semi-quantification of the bands are reported in figure matrix metallopeptidase, secreted protein, acidic, cysteine-rich and eno were secreted by hmads cells under all conditions, confirming the pattern of detection obtained by sds-page-mass spectrometry analysis. however, no significant difference was observed under the three conditions for these candidates. zymographic analysis of the secretion media revealed a prominent band of lysis at kda corresponding to the reported active form of mmp. addition of edta, an inhibitor of gelatinase activity, led to the absence of lytic bands, thus confirming the specificity of mmp activity. pigment epithelium derived factor was highly expressed in day adipocytes compared to day and day osteoblasts in agreement with mass spectrometry identification. for kda glucose-regulated protein precursor, transforming growth factor-beta-induced protein, pentraxin and pai- we confirmed the pattern of detection reported in table with significant differences of expression between day day adipocytes and day osteoblasts, as shown by western blot and histogram semi-quantifications. analysis of the plasminogen system in hmads cells differentiating towards adipocytes and osteoblasts given the pattern of expression at the early step of adipogenesis and osteogenesis of hmads cells, pai- appeared as one of the most promising candidates for investigating further the adipocyte/osteoblast balance. pai- mrna levels were similar to those of protein levels. since pai- is a key regulator of the conversion of plasminogen to plasmin, negatively acting on two plasminogen activators, namely upa and tpa, we decided to evaluate upa and tpa levels in hmads cell secretome. elisa experiments were performed on secretion media from hmads cells at day and day under adipogenic and osteogenic differentiating conditions after h of incubation. as reported in figure 5b, consistent with the immunoblotting data, pai- levels strongly decreased in differentiating adipocytes as compared to day by contrast, pai- was secreted by day osteoblasts, at albeit the same levels when compared to day concerning tpa measurement, we did not detect tpa in day differentiating adipocytes, while a significant increase of tpa levels was observed in day osteoblasts as compared to day. by contrast, upa remained undetectable in the secretion media of hmads cells under the three conditions analyzed. enzymatic activities of upa and tpa in h serum-free conditioned media of hmads cells at d and day of differentiating adipocytes and osteoblasts were then evaluated by casein/plasminogen zymographic analysis. as reported in figure 5d, tpa was mostly secreted by day osteoblasts, as observed by the lytic band at kda, while upa was undetectable, thus supporting elisa results. the higher band at around kda may correspond to complexes between plasminogen activators and pai- as classically reported. incubation of the gel with amiloride, which selectively abrogates upa-dependent enzyme activities, did not affect tpa-dependent lytic bands. furthermore, we demonstrated that hmads cells expressed upa receptor mrna as reported by semi-quantitative rt-pcr analysis. as these data actually suggest the involvement of plasminogen activation in the regulation of the fine balance between adipogenesis and osteogenesis, this hypothesis was next examined in a pathophysiological situation where such an imbalance occurs. pai-i expression in ovariectomized mice disequilibrium of the adipocyte/osteoblast balance is associated with the development of osteoporosis after ovariectomy. it is well established that in the bone of ovariectomized mice, adipocytes develop at expense of bone formation. in order to evaluate whether osteoporosis affects pai- in bone, we analyzed the expression of pai- mrna in the humerus of ovx compared to sham mice. ten weeks after surgery, as previously described mice increased fat mass as shown by leptin levels. as shown in figure 6b, the levels of pai- mrna decreased significantly in the humerus of ovx compared to sham mice, thus pointing out at pai- modulation in the adipocyte/osteoblast balance as a potential marker of osteoporosis. discussion characterization of hmads cell secretome the secretome of murine and human adipocytes has been characterized. however, these studies primarily focused on the late events of adipogenesis. concerning osteoblast secretome, a single detailed characterization has been performed so far by xiao and collaborators, who analyzed the extracellular matrix vesicle proteome of mineralizing osteoblasts and identified proteins at late steps of osteogenesis of mc3t3-e cells. herein, proteomic analysis has been used for the first time to compare the secretome of hmads cells differentiating into adipocytes and osteoblasts and to gain insights into the adipocyte/osteoblast balance. in order to delineate the initial secretory events of adipogenesis and osteogenesis, we performed a h short term secretion which likely limits the number of secreted proteins and cell lysis. a total of proteins was identified and classified into clusters. the largest cluster corresponds to extracellular matrix proteins, thus confirming the secretome characteristic of our fraction. in addition, more than 50% of hmads cell secretome is represented by proteins with signal peptide. cytoskeletal proteins and heat shock and folding proteins are also largely represented with and proteins, respectively. among the heat shock/folding proteins, two chaperones, e.g. hsp and hsp were found. these findings are reinforced by recent observations describing the presence of hsp and hsp in the bloodstream. hsp circulating levels in diabetic patients have been found associated to cardiovascular diseases, while elevated hsp circulating levels are related to systemic inflammatory reaction in patients with heart failure following acute myocardial infarction. in the metabolic enzyme cluster, enzymes were also found, including glyceraldehyde dehydrogenase and phosphoglycerate kinase. according to literature, extracellular gapdh, also found in rat serum, inhibits spreading of cos- cells, while pgk is secreted by tumor cells and its activity is regulated by hypoxia. these enzymes might reflect increased metabolic requirements, but they could also exert independent and still undiscovered functions. anti-oxidant proteins, like thioredoxin reductase, represent another cluster of proteins secreted by hmads cells. txnrd that does not exhibit a signal peptide, has been described in rat as a circulating protein. eleven percent of secreted proteins from hmads cells is however represented by intracellular proteins, presence of which has not been reported up to now outside the cells. a modest contamination by uncontrolled release of intracellular proteins cannot be excluded despite the fact that no cell lysis was detected. although cell death can take place in culture and is unavoidably accompanied by a subsequent release of cytosolic components, it should be pointed out that cell viability was not affected. among non-artifactual explanations, it is worth pointing out that this phenomenon could implicate exosome secretion, as most of the identified proteins were reported to be present in exosomes. exosomes are membrane vesicles that originate from the cell membrane and are released extracellularly. their occurrence has been also described in the bloodstream and in different tissues in vivo, suggesting their participation in physiological and/or pathological processes. to date, the exact function of exosomes in vivo is not completely understood. a large number of cell types, including reticulocytes, immune cells, platelets, epithelial cells, dendritic cells and others appears to secrete exosomes either as a mechanism to extrude some proteins and/or to communicate between cells and/or to play immune functions. typically, exosomes contain chaperones, cytoskeletal proteins, elongation factors and several enzymes, as described in literature through proteomic analysis of the secretion media from different sources as well as from analysis by flow cytometry and western blot. therefore intracellular proteins recovered outside hmads cells may be a mere reflection of events taking place within the cytosol at the time of exosome formation. furthermore, out of candidates, known to be intracellular, have a score higher than a fixed threshold when analyzed using software predicting a non-classical protein secretion, it is likely that these proteins are secreted by non-traditional mechanisms and did not arise from cell lysis. clearly, these proteins can only be considered as putatively secreted and their actual secretion should be assessed. selected candidates of hmads cell secretome mass spectrometry data have been validated for selected candidates. mmp sparc and eno displayed no significant differences in terms of quantification by immunoblotting or zymogram when comparing the three culture conditions. mmp a zinc-dependent endopeptidase, involved in the degradation of the ecm, is actively secreted by adipocytes and osteoblasts. the presence of mmp in hmads cells reinforces the concept that ecm remodelling represents a crucial event during the early steps of differentiation. as observed in this study, sparc has been already described as a secreted molecule in both adipocytes and osteoblasts. sparc is reported to be up-regulated in the adipose tissue of different models of murine obesity and its circulating levels correlate with body mass index in humans. a potential interest in further studying sparc in the adipocyte/osteoblast balance relies upon the fact that bone marrow stromal cells from sparc-null mice tend to form more adipocytes than cells from wild type mice. eno is mainly described for its role as an intracellular glycolytic enzyme, however its presence extracellularly has been already reported in the secretion medium of 3t3-l adipocytes, as well as in other cellular models. eno is a multifunctional protein and a putative plasminogen receptor, since it has been reported as a cell surface protein. therefore, a potential role of eno in the plasminogen cascade cannot be excluded. concerning the five other candidates, relevant differences of expression between day and day of adipogenesis and osteogenesis have been found. pedf has been reported to be preferentially secreted by 3t3-l preadipocytes as compared to fully differentiated adipocytes. however, zvonic et al. have described an increase in pedf expression during adipogenesis of primary cultures of human adipose-derived stem cells, in agreement with our observations in hmads cells when comparing hmads cells at day and after complete adipocyte differentiation. pedf could represent a valuable candidate for the adipocyte/osteoblast balance given its expression also in differentiating hmads osteoblasts. a putative role for pedf as mediator of angiogenesis and in matrix remodelling of the bone has been recently postulated. several molecular chaperones have been found in the secretome of hmads cells, including calumenin, calreticulin, protein disulfide isomerase heat shock proteins and grp as grp controls intracellular protein transport, its up-regulation in hmads differentiating adipocytes and osteoblasts as compared to day could reflect an increased demand in protein folding or a protection against endoplasmic reticulum stress. bigh is reported to be down-regulated during the differentiation of murine osteoblasts and to play negative effects on the early stages of osteogenesis. in hmads cells, bigh protein levels increase in differentiating osteoblasts as compared to day however a decrease during osteoblast terminal differentiation was observed. interestingly, bigh is also released by differentiating adipocytes at day so far, no report has described the secretion of bigh by adipocytes or adipose tissue, whereas bigh is involved in distinct cellular functions, such as cell growth, tumorigenesis, wound healing, apoptosis and migration. thus a role for bigh in adipogenesis remains to be established. ptx is a member of the pentraxin family that we described to be secreted by adipocytes and to respond to inflammatory stimuli such as tnfα. in differentiating hmads cells, ptx appears up-regulated during both adipogenesis and osteogenesis. the presence of ptx in osteoblasts has never been reported; we speculate that, under conditions of stress or inflammation, ptx could represent a potential target of the adipocyte/osteoblast balance that might deserve further studies. finally, pai- represents another member of the serpin family which is secreted by hmads cells. pai- is a pleiotropic molecule, exerting functional roles in wound healing, atherosclerosis, tumor angiogenesis, rheumatoid arthritis, fibrosis etc., besides its main role as regulator of fibrinolysis. in addition, pai- plays a role in metabolic disorders such as obesity and insulin resistance, representing a marker of metabolic syndrome. a role for pai- in bone remodeling has been postulated, since mice lacking pai- are protected from trabecular bone loss after ovariectomy, suggesting a site-specific role for pai- in bone turnover. altogether, these data indicate that pai- should represent a reliable candidate for the study of the adipocyte/osteoblast balance. in hmads cell secretome, pai- signal disappears at day of adipogenesis while remaining present in differentiating osteoblasts. despite some demonstration that pai- is produced by adipocytes during adipogenesis, it was recently shown that adipogenesis, as such, may not induce pai-1; rather it enhances the potential of adipocytes to respond to pai- inducers. this may explain why pai- synthesis dropped after differentiation of human adipocytes cultured in the absence of serum, known to contain several pai- inducers. moreover overexpression of pai- by adenovirus-mediated gene transfer inhibited adipocyte differentiation. conversely, preadipocytes from pai-1-/- mice showed greater differentiation than those issued from wild type mice. this suggests that not only pai- is dispensable for adipocyte differentiation but could even be deleterious as it has been recently suggested that bone marrow-derived pai- had an effect on the development of obesity through its effect in inflammation. tpa is also preferentially secreted by hmads differentiating osteoblasts, while no detectable levels of upa have been detected, despite the presence of upar mrna. interestingly, pai- and tpa serum levels have been reported as putative non-invasive diagnostic biomarkers of idiopathic osteonecrosis of the femoral head. as for circulating pai- a correlation with dysbaric osteonecrosis has been also described. these data strongly suggest that the plasminogen/plasmin system could represent a potential target for further investigation of the adipocyte/osteoblast balance. even if preliminary, our data concerning a decrease of pai- mrna levels in bone of ovx as compared to sham mice clearly indicate that pai- expression is modulated under conditions altering the adipocyte/osteoblast balance, such as osteoporosis.  in conclusion, we have characterized the secretome of hmads cells during the early events of adipogenesis and osteogenesis using a proteomic approach. hmads cell secretome is represented by bona fide secreted proteins, proteins secreted through non-classical pathways and intracellular proteins, the presence of which in the extracellular medium being likely due to exosome secretion. eight clusters have been identified, including proteases, protease inhibitors, ecm components, anti-inflammatory-antioxidant proteins, metabolic enzymes, cytoskeletal components, heat-shock/protein folding proteins and other proteins. among the selected proteins, pai- constitutes the most promising candidate for further studies owing to the role of the plasminogen/plasmin system in adipogenesis and bone remodeling. 
as secreted proteins could regulate the balance between both lineages, we aimed at characterizing the secretome of human multipotent adipose-derived stem cell at an early step of commitment to adipocytes and osteoblasts. it is well established that adipose tissue plays a key role in energy storage and release but is also a secretory organ and a source of stem cells. analysis of identified proteins showed that % corresponded to classical secreted proteins characterized by a signal peptide, that % previously described in the extracellular compartment were devoid of signal peptide and that % neither exhibited a signal peptide nor had been previously described extracellularly. a proteomic approach, using mono-dimensional electrophoresis and tandem mass spectrometry, allowed us to identify a total of proteins at day and day of adipocyte and osteoblast differentiation. given its regulation during the early events of hmads cell differentiation and its status in ovariectomized mice, pai- could play a role in the adipocyte/osteoblast balance and thus in bone diseases such as osteoporosis. among different lineages, stem cells are able to differentiate into adipocytes and osteoblasts. quantitative analysis has been performed for candidates: pai- pedf, bigh ptx sparc, eno grp and mmp among them, pai- was detected at day and day of osteoblast differentiation but never in adipocyte secretome.
 it is well established that adipose tissue plays a key role in energy storage and release but is also a secretory organ and a source of stem cells. among different lineages, stem cells are able to differentiate into adipocytes and osteoblasts. as secreted proteins could regulate the balance between both lineages, we aimed at characterizing the secretome of human multipotent adipose-derived stem cell at an early step of commitment to adipocytes and osteoblasts.  a proteomic approach, using mono-dimensional electrophoresis and tandem mass spectrometry, allowed us to identify a total of proteins at day and day of adipocyte and osteoblast differentiation. analysis of identified proteins showed that % corresponded to classical secreted proteins characterized by a signal peptide, that % previously described in the extracellular compartment were devoid of signal peptide and that % neither exhibited a signal peptide nor had been previously described extracellularly. these proteins were classified into clusters according to their function. quantitative analysis has been performed for candidates: pai- pedf, bigh ptx sparc, eno grp and mmp among them, pai- was detected at day and day of osteoblast differentiation but never in adipocyte secretome. furthermore we showed that pai- mrna was down-regulated in the bone of ovariectomized mice.  given its regulation during the early events of hmads cell differentiation and its status in ovariectomized mice, pai- could play a role in the adipocyte/osteoblast balance and thus in bone diseases such as osteoporosis. 