Fluid shear stress plays a critical role in vascular health and disease. While protein kinase A (PKA) has been implicated in shear-stimulated signaling events in endothelial cells, it remains unclear whether and how PKA is stimulated in response to shear stress. This issue was addressed in the present study by monitoring the phosphorylation of endogenous substrates of PKA. Shear stress stimulated the phosphorylation of cAMP responsive element binding protein (CREB) in a PKA-dependent manner. Western blot analysis using the antibody reactive against the consensus motif of PKA substrates detected two proteins, P135 and P50, whose phosphorylation was increased by shear stress. The phosphorylation of P135 was blocked by a PKA inhibitor, H89, but not by a phosphoinositide 3-kinase inhibitor, wortmannin. Expression of a constitutively active PKA subunit stimulated P135 phosphorylation, supporting the potential of P135 as a PKA substrate. P135 was identified as endothelial nitric oxide synthase (eNOS) by immunoprecipitation study. PKA appeared to mediate shear stress-stimulated eNOS activation. Shear stress stimulated intracellular translocation of PKA activity from 'soluble' to 'particulate' fractions without involving cellular cAMP increase. Taken together, this study suggests that shear stress stimulates PKA-dependent phosphorylation of target proteins including eNOS, probably by enhancing intracellular site-specific interactions between protein kinase and substrates.
Animals ; Aorta, Thoracic/cytology ; Blotting, Western ; Cattle ; Cell Culture Techniques ; Cell Extracts ; Cells, Cultured ; Comparative Study ; Cyclic AMP-Dependent Protein Kinases/analysis/*metabolism ; Endothelium, Vascular/cytology/*enzymology/*metabolism ; Nitric Oxide Synthase Type III/analysis/*metabolism ; Phosphorylation ; Precipitin Tests ; Research Support, Non-U.S. Gov't ; Stress, Mechanical ; Time Factors

Ultraviolet (UV) radiation-induced loss of dermal extracellular matrix is associated with skin photoaging. Recent studies demonstrated that keratinocyte-releasable stratifin (SFN) plays a critical role in skin collagen metabolism by inducing matrix metalloproteinase 1 (MMP1) expression in target fibroblasts. In the present study, we examined whether SFN released from UVB-irradiated epidermal keratinocytes increases MMP1 release from dermal fibroblasts, and whether these events are affected by p-coumaric acid (p-CA), a natural phenolic compound with UVB-shielding and antioxidant properties. HaCaT cells were exposed to UVB in the absence and presence of p-CA, and the conditioned medium was used to stimulate fibroblasts in medium transfer experiments. The cells and media were analyzed to determine the expressions/releases of SFN and MMP1. UVB exposure increased SFN release from keratinocytes into the medium. The conditioned medium of UVB-irradiated keratinocytes increased MMP1 release from fibroblasts. The depletion of SFN using a siRNA rendered the conditioned medium of UVB-irradiated keratinocytes ineffective at stimulating fibroblasts to release MMP1. p-CA mitigated UVB-induced SFN expression in keratinocytes, and attenuated the MMP1 release by fibroblasts in medium transfer experiments. In conclusion, the present study demonstrated that the use of UV absorbers such as p-CA would reduce UV-induced SFN-centered signaling events involved in skin photoaging.
Collagen ; Culture Media, Conditioned ; Extracellular Matrix ; Fibroblasts* ; Keratinocytes* ; Matrix Metalloproteinase 1* ; Metabolism ; Phenol ; RNA, Small Interfering ; Skin