The etiology and pathophysiologic process underlying AIS remains unclear despite the number of studies performed. Current views maintain that AIS is a multifactorial disease involving genetic 
, skeletal 
, environmental 
, biochemical 
, and neurohormonal factors 
It is generally recognized that abnormal growth is associated with the development and progression of the scoliotic curves 
. Furthermore, persistent general osteopenia of AIS patients indicates imbalance between bone resorption and bone formation in AIS 
. Interestingly, recent findings 
of diminished numbers of osteoblast and lower osteogenic differentiation abilities of MSCs from AIS patients rendered the MSCs impairment one possible mechanism of osteopenia in AIS, which has aroused growing concern.
The findings of abnormal bone growth and development in AIS, together with functional characteristics of MSCs, strongly suggest that MSCs may play a significant role in the etiology and pathogenesis of AIS. However, due to the lack of research in this area, we know very little of the biological characteristics, proteomic alterations, and the possible role of MSCs in the pathogenesis of AIS and accompanying generalized osteopenia. Therefore, to investigate the molecular mechanism of decreased osteogenic differentiation ability of MSCs and gain an insight into the pathogenesis of AIS, we employed 2D-DIGE and MS-based proteomic approaches to explore the differential protein expression patterns in MSCs of AIS and non-AIS controls. To the best of our knowledge, this study is the first research on AIS in the field of proteomics, and also one of only a few studies focused on MSCs from AIS patients.
As previously described, 41 spots were revealed with at least 1.3-fold changes in expression and 25 differentially expressed proteins were successfully identified by MALDI-TOF/TOF-MS. Due to their significant expression alterations as well as potential functional relevance to bone growth and development, five of these proteins, including PKM2, annexin A2, HSP27, γ-actin, and β-actin, were chosen to be further validated by Western blot analysis. These five differential proteins will be discussed in the following section.
It is well known that different isoenzymes of pyruvate kinase are expressed depending on the metabolic responsibilities of the various cells and tissues. Among them, pyruvate kinase isoenzyme type M2 (PKM2, M2-PK) is characteristic of cells with high rates of nucleic acid synthesis, including most of the proliferating cells, such as adult stem cells, embryonic cells, and tumor cells 
According to previous reports, PKM2 plays an important role in both cell proliferation and differentiation. In tumor cells, PKM2 has been regarded as an important metabolic sensor to adapt tumor metabolism to varying nutrient and oxygen supply conditions, thus facilitating cell proliferation and survival 
. It was also observed in the BB13 cell line that PKM2 translocated to the nucleus by IL-3 enhanced EGF-induced proliferation 
. In addition, using NIH3T3 cell line, Gilles A's finding suggested that PKM2 could regulate cell proliferation, cell growth and apoptosis in a glucose supply-dependent manner 
More importantly, inhibition of PKM2 induces a significant decrease in the population doubling (PDL) and cell proliferation rates, as well as an increase in cell size 
. Contrary to this, overexpression of PKM2 was found to enhance cell proliferation in the absence of interleukin-3 
. All of these studies suggest that PKM2 plays an important role in cell proliferation.
Furthermore, it has been reported that PKM2 is able to stimulate Oct-4-mediated transcriptional activation. Oct-4, as a central mediator of the undifferentiated pluripotent state of embryonic stem cells, may prevent expression of genes activated during differentiation 
. These observations indicate that PKM2 could negatively regulate cell differentiation through modulating the transactivation potential of the Oct-4 transcription factor.
In our study, PKM2 was significantly up-regulated in the MSCs of AIS, suggesting that the proliferation ability of MSCs in AIS might be increased. In contrast to our speculation, Park et al found the proliferation rate of MSCs obtained from AIS patients to be similar to that of the control subjects 
. However, the author also states that the number of patients included in their study was relatively small and suggests performing larger studies. Therefore, the exact alteration of MSC proliferation ability in AIS needs further large-scale studies to clarify.
Additionally, due to the negative correlation between PKM2 and cell differentiation, up-regulation of PKM2 in the AIS-MSCs in our experiment indicates decreased differentiation ability of MSCs in AIS, which not only support previous observation of reduction of osteogenic differentiation ability of AIS-MSCs, but also provide a possible mechanism of persistent general osteopenia of AIS patients.
In this study, annexin A2 was differentially down-regulated in MSCs obtained from AIS patients. These molecules belong to the annexin protein family, which have in common that they bind to acidic phospholipids in the presence of calcium 
. Annexin A2 contains four 70–80 amino acid repeats with an annexin consensus sequence. These four repeats form the conserved core region, which is responsible for the Ca2+
-dependent binding of the proteins to phospholipids 
Annexin A2, which is highly expressed by osteoblasts 
, osteoarthritic chondrocytes 
, hypertrophic and terminally differentiated growth plate chondrocytes 
, has been demonstrated to play a significant role in both intramembrane and intrachondral ossification.
In growth plate chondrocytes, it has been found that annexin A2 is essential to form Ca2+
channels in both the plasma membrane 
and matrix vesicles 
, which are particles released from the plasma membrane of mineralizing cells, and thereby initiating the mineralization process. The annexin-mediated alteration in Ca2+
homeostasis thereby regulates a whole sequence of events eventually leading to matrix mineralization. Furthermore, it has also been reported that inhibiting annexin channel function prevents terminal differentiation and the mineralization of growth plate chondrocytes in vitro 
Similarly, human osteoarthritic chondrocytes also release annexin A2-containing matrix vesicles, which initiate mineral formation 
. Annexin A2, which is not detectable in the upper, middle, and deep zones of healthy human articular cartilage, is expressed by chondrocytes in the upper zone of early- and late-staged human osteoarthritic cartilage 
Annexin A2 has also been shown to play an important role in the mineralization of osteoblastic cells. It was found that overexpression of annexin A2 led to increased ALP activity, which would be further elevated following differentiation 
. Since ALP has been considered a valuable indicator for bone development and differentiation, annexin A2 may alter ALP activity, thereby facilitating mineralization, a terminal step in the differentiation process of osteoblastic cells.
In studies of other cell types, there is much evidence to support the association between annexin A2 and cellular differentiation. For instance, annexin A2 expression is affected when myeloid cell lines are induced to differentiate by stimulation with all-trans-retinoic acid (ATRA) 
. As discussed above, annexin A2 may therefore be an important player in cellular differentiation and related disorders.
In this study, we demonstrated that annexin A2 was down-regulated significantly in MSCs from AIS patients, which was consistent with the previously described decrease in osteogenic differentiation ability. It is likely that annexin A2 plays an important role in osteogenic differentiation of MSCs from AIS patients and exerts further influence on both intramembrane and endochondral ossification in AIS. Therefore, annexin A2 might be responsible, at least in part, for the low bone mass in AIS, although the exact mechanism in etiology of AIS needs further research.
The mammalian small stress protein HSP27 (also denoted HSP28 and in murine cells, HSP25) belongs to the HSP family whose synthesis is induced or stimulated by heat shock and other forms of stress 
. We would like to discuss HSP27 from the following two aspects.
(1) HSP27 and Cell Differentiation
Recent studies have revealed that expression of the small heat shock (or stress) proteins (sHSP), including that of HSP27, is closely linked to changes in the state of cell differentiation. During the process of endochondral bone formation, sHSPs are differentially expressed in a stage-specific manner 
. Similar expression of sHSPs was observed during the differentiation of various mammalian cell types, such as embryonal carcinoma, embryonic stem cells 
, mouse Ehrlich ascites tumor cells 
, normal B cells, B lymphoma 
, osteoblasts, promyelocytic leukemia cells 
and NB4 promyelocytic cells 
In addition, transient accumulation of HSP27 has been observed during phorbol ester-induced monocytic differentiation of human HL-60 cells 
, as well as during ATRA-induced granulocytic differentiation of these cells 
In this study, HSP27 was down-regulated in MSCs from AIS patients. Together with the findings of previous studies, HSP27, as a mediator of cell differentiation, might be related to decreased differential ability of MSCs and clinical osteopenia in AIS patients.
(2) HSP27, HSP70 and Enviornmental Susceptibility
Many etiological studies of AIS suggest that idiopathic scoliosis is a genetic trait modified by environmental factors. Van Rhijn et al 
stated that differences in the development and progression of scoliosis, as well as age at detection (juvenile vs. adolescent) may be caused by environmental influences. In another study, van Rhijn et al 
noted that only half of twin pairs showed a difference in lateral Cobb angles of less than 10°, suggesting that curve severity may be affected by environment. In addition, Hermus 
reported a monozygotic twin pair that was described to be concordant for idiopathic scoliosis, but with different apical levels, magnitudes, and age at detection, further stressing the importance of environmental factors. However, the molecular mechanism of environmental susceptibility in AIS patients remains unclear.
Both HSP27 and HSP70 have been demonstrated as essential to ensure proper folding and intracellular localization of newly synthesized polypeptides 
. The expression of HSPs (including HSP27 and HSP70), which is activated by unfolded proteins, can enhance the cell's capacity not only to prevent protein aggregation and disassociate such aggregates once formed, but also to isolate such polypeptides in inclusions and selectively degrade them 
Since HSP27 and HSP70 can promote refolding, solubilization, and degradation of damaged polypeptides, the loss of this protective response should deteriorate the cell's capacity to handle the mutant or damaged proteins. In Parkinson's disease or Alzheimer's disease, for instance, such changes in the cell's proteolytic capacity, and the general increase in unfolded molecules, should further limit the capacity of the cell to deal with mutant proteins or other abnormal polypeptides and may thus indirectly contribute to the development of these disorders 
Our study showed down-regulation of both HSP27 and HSP70 in MSCs from AIS patients. If these proteins contribute a similar protective factor in AIS as in the diseases we discussed above, then its lower expression might subsequently compromise the capacity of MSCs to cope with aberrant polypeptides or damaged proteins caused by environmental pathogenic factors of AIS.
Taken together, it is tempting to speculate that the loss of these protective mechanisms, at least in part, renders AIS patients more susceptible to environmental factors, and thus eventually leads to the development and progression of scoliosis. The underlying mechanism of HSP27 and HSP70 in the etiology of AIS needs further elucidation.
β-actin and γ-actin
β-actin is usually found to be constitutively expressed, with the expression values often used for normalization of expression data. Interestingly, in our study, we found significantly decreased β-actin levels in MSCs from AIS patients.
It has been demonstrated that the actin cytoskeleton, consisting of both β-actin and γ-actin, changes from a large number of thin, parallel microfilament bundles extending across the entire cytoplasm in undifferentiated MSCs to a few, thick actin filament bundles located at the outermost periphery in MSC-differentiated osteoblasts 
. Therefore, it is postulated that the actin cytoskeleton may play a pivotal role in determining the hMSC mechanical properties and modulation of cellular mechanics during stem-cell osteodifferentiation 
During osteogenic differentiation, it was also observed that alterations in the cytoskeletal organization affect the expression of osteogenic differentiation markers, including alkaline phosphatase activity and calcium deposition 
. More interestingly, it was revealed that disrupting actin in hMSCs increased adipogenesis and decreased osteogenesis when compared to untreated controls, suggesting that the actin cytoskeleton might be important in the commitment process 
. In addition, it was reported that disruption of the actin cytoskeleton blocks osteoblastic differentiation of cells infected with constitutively active RhoA, which is one of the key regulators of cytoskeletal contractility 
Both β-actin and γ-actin were down-regulated in MSCs from AIS patients in our study, which was in agreement with previous reports of decreased osteogenic differentiation capacity of MSCs in AIS patients. Therefore, these data suggest that alteration of the actin cytoskeleton might be involved in the pathological mechanism of persistant general osteopenia in AIS. Additionally, it is worth mentioning that all five identified proteins in our experiment that were localized in cytoskeleton (β-actin, γ-actin, HSP27, WD repeat-containing protein 1, and moesin) were differentially down-regulated, indicating that disruption or inhibition of the cytoskeleton might contribute to the development and progression of AIS.
Down-regulation of β-actin, HSP27, γ-actin, and annexin A2, and up-regulation of PKM2, was reported for the first time as associated with the etiology of AIS in this experiment. Furthermore, the expression alterations of these five proteins indicate increased proliferation ability of MSCs and decreased osteogenic differentiation ability in AIS, the latter of which was also consistent with previous studies of AIS-MSC and might be one of mechanisms causing clinical osteopenia of AIS. In addition, the discovery of a total of 25 differentially expressed proteins in AIS patients may provide a valuable basis for further research on the characteristics of AIS-MSCs as well as the abnormal bone growth and development in AIS.
In summary, we have described the differential proteome of BM-MSCs from AIS patients for the first time. Our high-throughput proteomic approach based on 2D-DIGE technology followed by MS analysis has produced the differential proteome profile of BM-MSCs in AIS. A total of 25 proteins were identified as either up-regulated or down-regulated. These proteins might be involved in proliferation, differentiation, and other activities of MSCs. Furthermore, these differentially expressed proteins might play a significant role, in not only the causal mechanism of osteopenia in AIS, but also the AIS initiation and development. The identification of these proteins provides us important informations in understanding the underlying etiological mechanisms of AIS. Further studies are required to clarify the association between the possible changes of MSCs and the pathogenesis of AIS.