Isolation and expansion of adult HACs under physiological tonicity (380 mOsm) improves expression of chondrogenic markers on mRNA and protein levels. While other studies partially confirm that nonhuman chondrocytes respond to tonicity with altered aggrecan and SOX9
], we are reporting beneficial effects of isolating and expanding human normal and OA articular chondrocytes at physiological levels (380 mOsm). In addition, we also studied collagen type II expression, generally acknowledged to be the most important chondrogenic marker. As fibrocartilaginous collagen type I and hyaline collagen type II expression are differentially regulated in chondrocytes [34
], analyzing the collagen type II/type I expression ratios is informative of chondrogenic potential [51
]. Interestingly, NFAT5 seems to be crucially involved in this differential regulation upon hypertonic challenge: it positively regulates collagen type II, while suppressing collagen type I (Figure ). Fibrocartilage, occurring in areas subject to frequent stress like intervertebral discs and tendon attachment sites, is more rich in collagen type I than is hyaline cartilage [55
]. Tonicity may thus provide a simple means to manipulate expression of these two collagens for broader applications than regenerative chondrocyte implantations (autologous chondrocyte implantation or characterized chondrocyte implantation) alone [56
Under our conditions, COL2 mRNA abundances measured by quantitative PCR correlated well with protein synthesis as determined by Western blots (Figures and ). The same observation holds for COL1 expression in the early passages, but not for COL1 expression in the later passages.
Hypertonicity induced an increase in NFAT5 abundance, and protein synthesis rates were found to be proportional to the increase in mRNA in MDCK cells [28
] and mIMCD3 cells [27
mRNA is expressed abundantly in chondrocytes throughout passages and is further induced by hypertonicity. However, we failed to show NFAT5 protein expression by Western blotting. Whether this failure is due to low protein abundance in our cells or technical issues such as poor extraction efficiency of this very large transcription factor remains to be elucidated in future experiments.
Hypertonicity induces cell shrinkage, which may activate Na+
, or 2Cl-
co-transport, allowing cellular accumulation of NaCl and KCl. The beneficial effects on chondrogenic marker gene expression therefore could have been caused by accumulation of specific inorganic ions or specific channel activity rather than primarily tonicity-mediated effects. We used NMDG-Cl, a bulky substitute for small cations that is impermeable to almost all known channels [57
], and sucrose to exclude sodium-specific or chloride-specific effects. We were not able to detect any significant differences in gene expression patterns between the NaCl, NMDG-Cl or sucrose methods of tonicity alteration (data not shown).
As our initial studies concerned adult HACs obtained from OA knee joints, we aimed at eliminating interpretation bias due to the pathological state of these cells. Using identically challenged NHACs, we showed that these chondrocytes react similarly to the same order of tonicity with respect to our marker genes: 380 mOsm significantly delayed the phenotypical deterioration of NHACs as observed in control medium. This may imply that physiological tonicity, postulated to be around 380 mOsm for chondrocytes, is sensed by OA cells and normal cells in a similar fashion. We observed a slightly faster decrease in AGC1
mRNA levels in P2 and P3 NHACs as compared with OA HACs. Late-stage OA chondrocytes from fibrillated areas are dedifferentiated, flattened cells. The loss of a proper spherical shape as an integral part of the chondrocytes phenotype [58
] involves cytoskeletal changes [60
]. Exposing these cells to physiological tonicity as a redifferentiation stimulus probably induces a more enduring response as compared with spherical, normal chondrocytes. Cell-based therapies using the latter are usually restricted to younger individuals after traumatic insults. Autologous chondrocyte implantation employing OA cells may benefit relatively more from a hypertonic treatment protocol.
The precise molecular mechanism by which tonicity is sensed by cells is still poorly understood. Hypertonicity-increased NFAT5
mRNA abundances have been shown for other cell types [26
]. NFAT5 is thus accepted as key transcription factor participating in the mammalian hypertonic stress response. Our study is the first showing the functional expression of NFAT5
in HACs. In both OA and normal chondrocytes, cellular NFAT5
mRNA levels are increased by 380 mOsm. In addition, mRNA levels of the generally accepted NFAT5 target genes, S100A4
], were induced accordingly after hypertonic challenge, underscoring an involvement of NFAT5
. It has recently been suggested that guanine nucleotide exchange factors near the plasma membrane may be activated through cytoskeleton changes or by changes in interactions with putative osmosensors at the cell membrane in other cells [62
]. The sensation of such basic responses might not be different in chondrocytes than in other cells. Rho-type small G proteins [63
] and p38 kinases [64
] might also act upstream of NFAT5 in chondrocytes. In IMCD cells, p38 mitogen-activated protein kinase (MAPK) signaling was recently also shown to be involved in the NFAT5
-mediated hypertonic induction of the osmosensitive [66
] serine-threonine protein kinase Sgk-1 [68
]. As p38 MAPK plays important roles in chondrocytes and seems to be necessary for NFAT5
], further experiments employing pharmacological inhibition or knockdown experiments in HACs will hopefully shed more light into this signaling cascade in chondrocytes.
An increase in NFAT5 mRNA is usually transient with a cell type-dependent time course and a twofold to fourfold upregulation [26
], which fits with our data. NFAT5
mRNA abundance might rapidly increase upon hypertonic stress by a transient increase in its mRNA stability, mediated by its 5'-untranslated region [27
]. Whether 380 mOsm is a sufficiently high tonicity to explain our increase in mRNA by this phenomenon, or whether active transcription is involved, has to be addressed in other studies. Interestingly, Tew and colleagues showed very recently that the mRNA of SOX9
, an important regulator of COL2 expression, is stabilized by supraphysiological tonicity [70
]. Therefore, 380 mOsm might also directly contribute to SOX9
mRNA stability and abundance in our experiment, rather than elevating promoter activity. COL2 regulation could thus be an indirect effect of tonicity.
Interestingly, AGC1 seems to be more stably expressed in cultures maintained at 280 mOsm compared with 380 mOsm, with a lower overall expression in the former condition. Effects of tonicity on promoter activity and mRNA stability of AGC1
are incompletely understood. Other groups have described the complexity of osmotic stress on gene expression [71
]. It is tempting to speculate that gene expression may be influenced by morphological changes between our conditions: while cells cultured at 380 mOsm are rather round, cells cultured in monolayer at 280 mOsm are rather flat and more fibroblast-like (see Figure ). Although we did not investigate actin stress fiber formation in the present study, they are usually more pronounced in fibroblastic cells and have been shown to suppress SOX9
mRNA levels in chondrocytes [50
Aggrecan expression, however, has been reported to be influenced by both hypertonicity and hypotonicity [4
]. The promoter regions of both collagen type II and AGC1
contain a plethora of potential other binding sites for transcriptional enhancers and suppressors, such as SOX5/6 [73
], Barx2 [75
], β-catenin [76
], c-Maf [77
], PIAS [78
], TRAP230 [79
], Bapx1 [80
], and C/EBP and NF-κB [81
]. Chondrogenic differentiation and the SOX9 dependency of aggrecan and collagen expression may also be differentially modulated by these transcriptional cofactors under different tonicities. Interestingly, while the SOX9 dependency of COL2A1 expression has been unequivocally shown, it may not actually be a key regulator of COL2A1
promoter activity in human adult articular chondrocytes [82
]. Of note, the human aggrecan promoter sequence has been shown to contain a conserved NFAT5 binding site [83
]. In nucleus pulposus cells, SOX9-mediated aggrecan expression has recently been shown to critically depend on PI3K/AKT signaling [84
]. Moreover, while high NaCl rapidly activates p38 MAPK, its action can be isoform specific and may exert opposing effects on NFAT5 [85
], which in turn may influence COL2A1
transcription differently in a tonicity-dependent manner. We are therefore currently looking into the underlying molecular mechanisms regulating AGC1 and COL2 expression in both conditions.
With respect to regenerative medical applications, the high-end hypertonic conditions used by Tew and colleagues can be considered a limitation of that study. In our hands, these tonicity levels (≥ 480 mOsm) induced chondrocyte death within 48 hours (Figure ) and are probably not applicable for chondrocyte expansion culture. To ensure sufficient cell numbers for cell-based repair techniques, the proliferation capacity of the isolated chondrocytes should not be compromised. Cell numbers generally need to be increased during two passages (>4 to 10 times) for clinical application [86
]. We found that supraphysiological conditions (480 mOsm and 580 mOsm) clearly compromised survival rates, which is in agreement with data by Racz and colleagues [17
]. From our data, we conclude that about 380 mOsm is optimal for both isolation and in vitro
expansion culture of HACs.
NFAT5 knockdown downregulates its own transcription by 75% and compromises target gene induction (Figure ), being in line with functionally active NFAT5 in chondrocytes. Constitutive homodimeric NFAT5 molecules encircle DNA rather independently of tonicity in solution [88
], enabling NFAT5 to exert its biological activity over a wide tonicity range [89
]. It is thus reasonable to assume that NFAT5 activity is not generally compromised at 380 mOsm. However, other aspects are involved in the regulation of NFAT5 as well as its target genes. Like other proteins larger than 50 kDa [91
], NFAT5 depends on nuclear localization and export sequences for its nuclear translocation [26
]. In most cells, NFAT5 is equally distributed between the cytoplasm and the nucleus at physiological tonicity (± 300 mOsm), whereas at 500 mOsm most of it localizes to the nucleus [19
To demonstrate that the hypertonicity-induced chondrogenic marker expression was indeed mediated by NFAT5, we used RNAi to confirm that knockdown of NFAT5 significantly inhibited hypertonic induction of its own transcription as discussed before, significantly suppressed the tonicity-mediated induction of known NFAT5 targets, and, most importantly, significantly eliminated the hypertonicity-mediated mRNA expression of chondrogenic marker genes (COL2, AGC1, SOX9 and COL1).