Jeong Soon Lee

Mechanical stretch suppresses BMP4 induction of stem cell adipogenesis via upregulating ERK but not through downregulating Smad or p38

Bone morphogenetic proteins (BMPs) are also implicated in the commitment of mesenchymal stem cells (MSCs) toward adipocytes. We tested that stretching of cells may downregulate BMP4 induction of MSC adipogenesis. C3H10T1/2 MSCs were pretreated with BMP4 and induced to differentiate to adipocytes using adipogenic hormonal inducers. To test the stretch effect on BMP4 function, cells were exposed to cyclic tensile stretch (10% strain, 0.25Hz, 120min/day) during the BMP4 pretreatment period. BMP4 induced MSC adipocytic commitment. Stretching during the BMP4 exposure could suppress BMP4 induction of MSC adipogenesis, as assessed by downregulated adipogenic transcription factors (PPARγ, C/EBPα, aP2) and decreased lipid accumulation. BMP4 signaled through Smad1/5/8 and p38MAPK, whereas cell stretch did not affect BMP4-induced activation in Smad or p38. On the other hand, cell stretch triggered significant ERK1/2 phosphorylation relative to BMP4 treatment alone cells. Further, stretch suppression of BMP4-induced MSC adipogenesis was significantly deteriorated if cells were stretched with ERK blocked by PD98059. Combined, these suggest that cell stretch suppresses the BMP4 induction of MSC adipogenesis potentially via upregulating ERK but not through the downregulation of Smad or p38. Our data on inhibiting MSC adipogenesis will be of significant interest for obesity and developmental mechanobiology studies.

Retinoic acid inhibits BMP4-induced C3H10T1/2 stem cell commitment to adipocyte via downregulating Smad/p38MAPK signaling.

Increased adipocyte formation from mesenchymal stem cells (MSCs) is typical for obesity. It is recently observed that bone morphogenetic proteins (BMPs) provide instructive signals for the commitment of MSCs to adipocytes. We examined potential role of retinoic acid (RA) in inhibiting the BMP4 induction of MSC commitment toward adipocyte. BMP4-treated C3H10T1/2 MSCs, when further exposed to adipogenic differentiation media, displayed distinct adipocytic commitment and differentiation. This could be inhibited by RA exposure during the BMP4 treatment stage (commitment stage before adipogenic hormonal inducers were given), as was observed by reductions in key adipogenic genes/transcription factors (C/EBPα, PPARγ, aP2), lipogenic genes (LPL, FAS, GLUT4), and lipid accumulation. Among RA receptors (RARs) screened, RARβ was mainly upregulated under RA exposure. BMP4 signaled through both Smad1/5/8 and p38 mitogen-activated protein kinase (MAPK) and RA significantly suppressed the BMP4-triggered phosphorylation of both Smad1/5/8 and p38MAPK. These data suggest that RA has inhibitory effects on the BMP4 induction of C3H10T1/2 adipocytic commitment via downregulating Smad/p38MAPK signaling. How to inhibit MSC adipocytic commitment, as partly revealed in this study, will have a significant impact on treating obesity and related diseases.

The role of RhoA kinase (ROCK) in cell alignment on nanofibers

While the potential of nanofibers as tissue engineering scaffolds has been demonstrated, very little has been revealed as regards the molecular mechanism by which cells sense and respond to nanofibers. It was hypothesized that RhoA kinase (ROCK), one of the vital cell tension signaling cascades, plays a role in regulating cell alignment on nanofibers. To test this, unidirectionally aligned and randomly distributed nanofibers, both with an average diameter of ∼130nm, were fabricated with poly(l-lactic acid) (PLLA). A flat PLLA film was used as the control. Mesenchymal stem cells (MSCs, C3H10T1/2) displayed high fidelity in cell orientation along aligned nanofibers, and showed an increased cell spreading area on random nanofibers. Interestingly, cells cultured on aligned nanofibers displayed significantly greater ROCK expression relative to cells on a flat surface, as assessed by immunoblotting. To further test the role of ROCK, MSCs with ROCK small hairpin RNA (shRNA) were established. It is notable that, even when ROCK was stably knocked down via shRNA, cells could still display preferred orientation along aligned nanofibers. However, MSCs with shRNA-ROCK displayed a significantly decreased cell major axis length following aligned nanofibers compared with shRNA vector control, suggesting that ROCK may be involved in cell elongation on aligned nanofibers. Along with the reduction in cell length, cell area was decreased with ROCK silencing. These cell morphological changes induced by shRNA-ROCK were generally maintained on a flat surface and random nanofibers. A pharmacological ROCK inhibitor, Y-27632, produced results similar to those of shRNA-ROCK. The data on the role of ROCK in regulating cell alignment on nanofibers may provide a new mechanistic insight into nanofiber control of cells.

Inducing neurite outgrowth by mechanical cell stretch.

Abstract Establishing extracellular milieus to stimulate neuronal regeneration is a critical need in neuronal tissue engineering. Many studies have used a soluble factor (such as nerve growth factor or retinoic acid [RA]), micropatterned substrate, and electrical stimulation to induce enhanced neurogenesis in neuronal precursor cells. However, little attention has been paid to mechanical stimulation because neuronal cells are not generally recognized as being mechanically functional, a characteristic of mechanoresponsive cells such as osteoblasts, chondrocytes, and muscle cells. In this study, we performed proof-of-concept experiments to demonstrate the potential anabolic effects of mechanical stretch to enhance cellular neurogenesis. We cultured human neuroblastoma (SH-SY5Y) cells on collagen-coated membrane and applied 10% equibiaxial dynamic stretch (0.25 Hz, 120 min/d for 7 days) using a Flexcell device. Interestingly, cell stretch alone, even without a soluble neurogenic stimulatory factor (RA), produced significantly more and longer neurites than the non–RA-treated, static control. Specific neuronal differentiation and cytoskeletal markers (e.g., microtubule-associated protein 2 and neurofilament light chain) displayed compatible variations with respect to stretch stimulation.

Micropatterning-retinoic acid co-control of neuronal cell morphology and neurite outgrowth

Creating physical-biochemical superposed microenvironments optimal for stimulating neurite outgrowth would be beneficial for neuronal regenerative medicine. We investigated potential co-regulatory effects of cell micropatterning and retinoic acid (RA) soluble factor on neuronal cell morphology and neurite outgrowth. Human neuroblastoma (SH-SY5Y) cell patterning sensitivity could be enhanced by poly-L-lysine-g-polyethylene glycol cell-repellent back-filling, enabling cell confinement in lanes as narrow as 5 μm. Cells patterned on narrow (5 and 10 μm) lanes showed preferred nucleus orientation following the patterning direction. These cells also showed high nucleus aspect ratio but constrained nucleus spreading. On the other hand, cells on wide (20 μm and above) lanes showed random nucleus orientation and cell and nucleus sizes similar to those on unpatterned controls. All these changes were generally maintained with or without RA. Confining cells on narrow (5 and 10 μm) lanes, even without RA, significantly enhanced neurite extension relative to unpatterned control, which was further stimulated by RA. Interestingly, cell patterning on 5 and 10 μm lanes without RA produced longer neurites relative to the RA treatment alone case. Our data on the potential interplay between microscale physical cell confinement and RA-soluble stimulation may provide a new, integrative insight on how to trigger neurite/axon formation for neuronal regenerative medicine.

Graphene substrate for inducing neurite outgrowth.

A few recent studies demonstrated that graphene may have cytocompatibility with several cell types. However, when assessing cell behavior on graphene, there has been no precise control over the quality of graphene, number of graphene layers, and substrate surface coverage by graphene. In this study, using well-controlled monolayer graphene film substrates we tested the cytocompatibility of graphene for human neuroblastoma (SH-SY5Y) cell culture. A large-scale monolayer graphene film grown on Cu foils by chemical vapor deposition (CVD) could be successfully transferred onto glass substrates by wet transfer technique. We observed that graphene substrate could induce enhanced neurite outgrowth, both in neurite length and number, compared with control glass substrate. Interestingly, the positive stimulatory effect by graphene was achieved even in the absence of soluble neurogenic factor, retinoic acid (RA). Key genes relevant to cell neurogenesis, e.g., neurofilament light chain (NFL), were also upregulated on graphene. Inhibitor studies suggested that the graphene stimulation of cellular neurogenesis may be achieved through focal adhesion kinase (FAK) and p38 mitogen-activated protein kinase (MAPK) cascades. Our data indicate that graphene may be exploited as a platform for neural regenerative medicine, and the suggested molecular mechanism may provide an insight into the graphene control of neural cells.

The role of focal adhesion kinase in BMP4 induction of mesenchymal stem cell adipogenesis.

Obesity is characterized by excessive adipocytic number growth and resultant adipose tissue hyperplasia. However, molecular mechanisms of abnormal recruitment of new adipocytes from precursor cells are not fully known. Several studies showed that bone morphogenetic proteins (BMPs) also play a role in inducing mesenchymal stem cells (MSCs) to commit to adipocytes. We tested the hypothesis that focal adhesion kinase (FAK), one of the vital focal adhesion signaling molecules, is required for BMP4 induction of MSC adipogenesis. BMP4 exposure triggered FAK activation at pY397 auto-phosphorylation site in murine C3H10T1/2 MSCs. Interestingly, silencing FAK by small hairpin RNA (shRNA) significantly suppressed BMP4 induction of MSC adipogenic activities, including lipid accumulation and expression of key adipogenic genes (C/EBPα, PPARγ, aP2), as relative to shRNA vector control. As a potential molecular mechanism, BMP4-triggered phosphorylation in Smad1/5/8 and p38 was significantly downregulated by shRNA-FAK. Pharmacological FAK inhibitor 14 provided similar results in BMP4-mediated MSC adipogenesis and Smad/p38 signaling. Our data clearly suggest a link between FAK and BMP4 induction of MSC adipogenesis, and may indicate a potential therapeutic approach targeting FAK for dealing with obesity.

Mechanical Control of Mesenchymal Stem Cell Adipogenesis

Increased Mesenchymal Stem Cell (MSC) commitment and differentiation into adipocytes contributes to obesity. Other than dietary and biochemical factors, recent studies have begun to explore the role of mechanical signals in controlling MSC adipocytic commitment and differentiation. Several reported data suggest that by subjecting MSCs to certain mechanical stimuli, such as stretching, compression, and fluid shear, their adipogenesis could be inhibited or decreased. However, it is still very early to draw conclusions on the detailed mechanical regimens to optimally inhibit MSC adipogenesis and on the molecular pathways governing such adipogenesis-inhibitory mechanosensitive signaling. In this commentary, key data on the mechanical control of MSC adipogenesis and proposed molecular mechanisms will be highlighted and a future perspective in this new topic area will be provided.

Flowtaxis of osteoblast migration under fluid shear and the effect of RhoA kinase silencing.

Despite the important role of mechanical signals in bone remodeling, relatively little is known about how fluid shear affects osteoblastic cell migration behavior. Here we demonstrated that MC3T3-E1 osteoblast migration could be activated by physiologically-relevant levels of fluid shear in a shear stress-dependent manner. Interestingly, shear-sensitive osteoblast migration behavior was prominent only during the initial period after the onset of the steady flow (for about 30 min), exhibiting shear stress-dependent migration speed, displacement, arrest coefficient, and motility coefficient. For example, cell speed at 1 min was 0.28, 0.47, 0.51, and 0.84 μm min-1 for static, 2, 15, and 25 dyne cm-2 shear stress, respectively. Arrest coefficient (measuring how often cells are paused during migration) assessed for the first 30 min was 0.40, 0.26, 0.24, and 0.12 respectively for static, 2, 15, and 25 dyne cm-2. After this initial period, osteoblasts under steady flow showed decreased migration capacity and diminished shear stress dependency. Molecular interference of RhoA kinase (ROCK), a regulator of cytoskeletal tension signaling, was found to increase the shear-sensitive window beyond the initial period. Cells with ROCK-shRNA had increased migration in the flow direction and continued shear sensitivity, resulting in greater root mean square displacement at the end of 120 min of measurement. It is notable that the transient osteoblast migration behavior was in sharp contrast to mesenchymal stem cells that exhibited sustained shear sensitivity (as we recently reported, J. R. Soc. Interface. 2015; 12:20141351). The study of fluid shear as a driving force for cell migration, i.e., “flowtaxis”, and investigation of molecular mechanosensors governing such behavior (e.g., ROCK as tested in this study) may provide new and improved insights into the fundamental understanding of cell migration-based homeostasis.