To better understand the role of mechanical and environmental factors in adipogenesis, we have examined how altered cell morphology (and the associated decrease in cytoskeletal tension) may synergistically interact with physiological oxygen conditions to regulate adipogenic differentiation of ASCs. In this study, we demonstrated a significant influence of cytoskeletal organization on adipogenic differentiation under both physiological and ambient oxygen conditions. Treatment with cytoskeletal inhibitors to reduce apparent cytoskeletal tension in primary adult human ASCs enhanced the expression of adipogenic marker genes as well as functional indicators of lipid metabolism.
Combining physiological oxygen and cytoskeletal inhibition had a dramatic impact on ASC adipogenesis. Under physiological oxygen tension with cytoskeletal inhibition, PPARγ and FABP4 gene expression levels were significantly increased following seven days of induction compared to ambient oxygen and non-inhibited controls (Figure 3). These trends were maintained following 21 days of induction. Exposure to physiological oxygen tension for 21 days significantly down-regulated FABP4 gene expression, but cytoskeletal inhibition reversed this decrease (Figure 3). Physiological oxygen tension also significantly decreased GPDH activity (Figure 5) and triglyceride accumulation (Figure 6). These decreases in functional markers of mature adipocytes may be related to the diminished FABP4 gene expression.
Based on our results, we postulate that within the in vivo ASC niche at physiological oxygen levels, cytoskeletal tension may regulate adipose tissue expansion. The combined effect of cytoskeletal tension and physiological oxygen levels on ASCs has not been previously reported. Our observations on the effects of either cytoskeletal inhibition or reduced oxygen tension, applied individually, are qualitatively consistent with previously published studies using other types of stem cells. Reducing cytoskeletal tension via cytochalasin D and blebbistatin induced rounded cell morphology and up-regulated adipogenesis in MSCs  and C3H10T1/2 cells, an immortalized MSC line . In vivo and in vitro, preadipocytes possess a fibroblastic morphology; however, mature adipocytes are more spherical [8, 11]. The mechanisms driving the morphological change during adipogenesis in vivo remain unclear. In the present study, we chose to induce these changes in vitro through the use of chemical inhibitors that target intracellular cytoskeletal tension. While cytochalasin D and blebbistatin act through different mechanisms to disrupt the actin cytoskeleton, they produced similar cell shape changes and enhanced adipogenesis, suggesting that the enhancement is due to actin disassembly and reorganization.
Physiological oxygen tension, which is substantially lower than the ambient oxygen tension typically used in cell culture studies, has been shown to inhibit stem cell differentiation and aid in maintaining pluripotency through hypoxia-inducible factor (HIF)-regulated pathways . Previous in vitro adipogenesis studies using low oxygen conditions or chemical treatments mimicking hypoxia have demonstrated that low oxygen ten-sion inhibits or reduces adipogenic differentiation in preadipocyte cell lines [6, 17, 18]. Studies with primary stem cells found that low oxygen conditions not only reduced lineage commitment in ASCs , but extended the differentiation potential in adipose tissue stromal cells  and bone marrow-derived MSCs  compared to ambient oxygen conditions. Additionally, when cultured under low oxygen, human MSCs showed diminished adipogenic differentiation and FABP4 gene expression . Similar to the MSCs, we demonstrated suppression of adipogenic differentiation marker expression (FABP4, GPDH and triglycerides) in the ASCs under physiological oxygen conditions. In contrast, ASCs cultured under chondrogenic conditions and 5% O2 significantly increased cartilaginous matrix production while cell proliferation decreased . Taken together, these and our findings demonstrate that reducing the oxygen tension to a physiological level can differentially regulate the switch between growth and differentiation in ASCs.
The FABP4 gene encodes an intracellular fatty acid binding protein that is found in differentiated adipocytes . During adipogenesis, the adipogenic transcription factors PPARγ and CCAAT/enhancer binding protein α (C/EBPα) induce FABP4 mRNA expression, resulting in increased FABP4 protein synthesis . Studies with FABP4-deficient mice revealed that FABP4 plays a role in adipocyte fatty acid metabolism, triglyceride storage and insulin resistance . Our results showed that low oxygen tension reduced FABP4 gene expression relative to ambient oxygen tension in ASCs at 21 days (Figure 3). This was consistent with an earlier study involving MSCs , though the exact mechanism for FABP4 reduction was not examined. We did not find a corresponding decrease in PPARγ, which typically regulates FABP4 expression, suggesting that FABP4 expression may also be controlled through a different mechanism. The ASCs with reduced FABP4 expression also had significantly decreased triglyceride content and GPDH activity, suggesting that lipid synthesis and storage can also respond to lowered oxygen tension via a PPARγ-independent mechanism. Interestingly, cytoskeletal inhibition abolished the physiological oxygen-mediated down-regulation of adipogenic markers, suggesting that cytoskeletal tension may act as an overriding regulator of ASC-to-adipocyte differentiation in vivo.
Our results indicate there may be significant crosstalk between the oxygen tension and cytoskeletal pathways to regulate adipogenesis, though specific signaling pathways have yet to be elucidated. Unfortunately, current literature does not provide a clear picture of how oxygen tension and cytoskeletal tension pathways interact. Several pathways regulate adipogenesis via cytoskeletal tension, including the RhoA/Rho-associated protein kinase (ROCK) pathway. ROCK is capable of phosphorylating several molecules, including myosin light chain and myosin light chain phosphatase. The phosphorylation of these molecules increases non-muscle myosin type II activity, which regulates the tension between actin microfilaments. Several studies have shown that RhoA/ROCK-mediated cytoskeletal tension can be a key regulator of adipogenesis in stem cells [14, 15]. Treatment of the cells with cytochalasin D or blebbistatin led to an up-regulation of adipogenic gene expression (Figure 3) and an increase in GPDH activity (Figure 5) and triglyceride content (Figure 6) suggesting that the RhoA/ROCK pathway could be involved. However, the previous studies on cytoskeletal tension and adipogenesis were conducted under ambient oxygen conditions and did not address the effects that low oxygen tension would have on the differentiation process. The few studies that did examine how the RhoA/ROCK pathway functions under low oxygen suggest that RhoA activation induces HIF-1α expression . HIF-1α is regarded as the primary transcription factor of hypoxia-regulated functions and has been linked to the down-regulation of adipogenesis in vitro by inhibiting the transcription of C/EBPβ and PPARγ in 3T3-L1 preadipocytes [6, 18]. However, the studies linking HIF-1α and RhoA did not indicate whether downstream components of the RhoA/ROCK pathway, specifically, cytoskeletal tension, were involved in regulating HIF-1α .
A potential limitation of this study is the use of small molecule chemical inhibitors to disassemble the actin microfilaments (via cytochalasin D) or to inhibit non-muscle myosin type II binding to actin (via blebbistatin) to reduce cytoskeletal tension and alter cell shape. These inhibitors affect the cell morphology leading to cell rounding, whereas cellular shape may also be regulated by extracellular matrix factors, such as substrate stiffness. Exploring external mechanical factors with an intact actin cytoskeleton may provide additional insights into how cell shape influences and is controlled during adipogenesis in vivo. ASCs and mature adipocytes occupy unique extracellular niches within the adipose tissue environment , and differences between niche mechanical properties could have a significant impact on actin cytoskeleton assembly, morphology and mechanotransduction. For instance, ASCs cultured on a soft substrate demonstrated an amorphous actin cytoskeleton and rounded morphology, compared to the pronounced actin filaments and fibroblastic morphology associated with stiff substrates . Our current study lays the foundation for investigating synergetic effects of the cytoskeleton and oxygen tension. Future studies will focus on regulating the actin cytoskeleton through other means such as matrix stiffness, which has previously been shown to influence adipogenesis . A second potential limitation of this study and nearly all in vitro adipogenic differentiation studies is the use of synthetic chemical cocktails to induce differentiation. This methodology is well established to study stem cell biology, but is likely not entirely representative of in vivo differentiation conditions. Therefore, we must use caution when extrapolating in vitro results to in vivo differentiation. Additionally, this study was conducted in a typical two-dimensional monolayer culture, making the results easier to compare with prior studies. Future studies are warranted to conduct experiments in three-dimensional culture systems that more closely mimic the in vivo microenvironment.