The regulation of β-adrenergic receptor-mediated PKA activation by substrate stiffness via microtubule dynamics in human MSCs

The mechanical microenvironment surrounding cells has a significant impact on cellular function. One prominent example is that the stiffness of the substrate directs stem cell differentiation. However, the underlying mechanisms of how mechanical cues affect stem cell functions are largely elusive. Here, we report that in human mesenchymal stem cells (HMSCs), substrate stiffness can regulate cellular responses to a β-adrenergic receptor (β-AR) agonist, Isoproterenol (ISO). Fluorescence resonance energy transfer-based A-Kinase Activity Reporter revealed that HMSCs displayed low activity of ISO-induced protein kinase A (PKA) signal on soft substrate, whereas a significantly higher activity can be observed on hard substrate. Meanwhile, there is an increasing ISO-induced internalization of β₂-AR with increasing substrate stiffness. Further experiments revealed that the effects of substrate stiffness on both events were disrupted by interfering the polymerization of microtubules, but not actin filaments. Mechanistic investigation revealed that inhibiting ISO-induced PKA activation abolished β₂-AR internalization and vice versa, forming a feedback loop. Thus, our results suggest that the cellular sensing mechanism of its mechanical environment, such as substrate stiffness, affects its response to chemical stimulation of β-AR signaling and PKA activation through the coordination of microtubules, which may contribute to how mechanical cues direct stem cell differentiation.