Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase which participates in many important cellular processes such as cell adhesion and migration. However, the role of FAK in renal tubular epithelial-to-mesenchymal transition (EMT) is still unknown. FAK was knocked down by transfection of specific small interfering RNA (siRNA) in cultured HK-2 cells, then the cells were stimulated with transforming growth factor-beta 1 (TGF-beta1). The expression of FAK, alpha-smooth muscle actin (alpha-SMA),E-cadherin, Akt, matrix metallopeptidase-9 (MMP-9),tissue inhibitor of metalloproteinase-1 (TIMP-1), and collagen IV were detected by RT-PCR, Western blot and immunofluorescence methods, respectively. Cell migration was determined by transwell assay. The results suggest that the expression of FAK was up-regulated in HK-2 cells when incubated with TGF-beta1(10 microg/l), which was accompanied by reduced expression of E-cadherin and increased expression of alpha-SMA. All these changes were restored by FAK siRNA. Akt phosphorylation was induced by the treatment with TGF-beta1, which was blocked by FAK siRNA. TGF-beta1-induced down-regulation of E-cadherin was recovered by a PI3K/Akt inhibitor, LY294002, without affecting the expression of FAK. Functionally, TGF-beta1 induced an increase in MMP-9 expression, as well as decreased expression of TIMP-1 and collagen IV, which were all restored by the FAK siRNA transfection. In addition, FAK siRNA significantly reduced TGF-beta1-induced cells migration. In conclusion, FAK may play a crucial role in mediating TGF-beta1-induced EMT through the activation of Akt pathway.

In studying the differentiation of stem cells along smooth muscle lineage, smooth muscle cell (SMC) contractile proteins serve as markers for the relative state of maturation. Yet, recent evidence suggests that some SMC markers are probably expressed in multipotent mesenchymal stem cells (MSCs). Such a paradox necessitates investigations to re‐examine their role as differentiated markers in MSCs. We tried to detect the expression of four widely used SMC markers including α‐smooth muscle actin (α‐SMA), h1‐calponin, desmin and smooth muscle myosin heavy chain (SM‐MHC), as well as the other isoforms of calponin family in resting MSCs. Then we used three different conditions to initiate MSCs differentiation along SMC lineage, and examined the alternation of SMC markers expression at both the transcript level and protein level. Desmin and h1‐calponin are expressed in MSCs, in the presence or absence of SMC induction conditions. Moreover, MSCs are shown to express all known isoforms of calponin. Double‐staining reveals that h1‐calponin +/α‐SMA – cells constitute the majority of resting MSCs. Under differentiated conditions, expression of SM‐MHC was initiated and expression of α‐SMA was promoted. The expression of SM‐MHC and upregulation of α‐SMA are relatively reliable indications of a mature smooth muscle phenotype in MSCs. Given that the cells are particularly rich in calponins expression, we postulate possible roles of these proteins in regulating cellular function by taking part in actin cytoskeleton and signaling. These findings imply that an extensive study of the cell physiology of MSCs should focus on the functional roles for these proteins, rather than simply regard them as differentiated markers.

BackgroundIn murine heart failure models and in humans with diabetic‐related heart hypertrophy, inhibition of phosphodiesterase 5 (PDE5) by sildenafil improves cardiac outcomes. However, the mechanism by which sildenafil improves cardiac function is unclear. We have observed a relationship between PDE5 and β2 adrenergic receptor (β2AR), which is characterized here as a novel mechanistic axis by which sildenafil improves symptoms of diabetic cardiomyopathy.Methods and ResultsWild‐type and β2AR knockout mice fed a high fat diet (HFD) were treated with sildenafil, and echocardiogram analysis was performed. Cardiomyocytes were isolated for excitation‐contraction (E‐C) coupling, fluorescence resonant energy transfer, and proximity ligation assays; while heart tissues were implemented for biochemical and histological analyses. PDE5 selectively associates with β2AR, but not β1 adrenergic receptor, and inhibition of PDE5 with sildenafil restores the impaired response to adrenergic stimulation in HFD mice and isolated ventriculomyocytes. Sildenafil enhances β adrenergic receptor (βAR)‐stimulated cGMP and cAMP signals in HFD myocytes. Consequently, inhibition of PDE5 leads to protein kinase G–, and to a lesser extent, calcium/calmodulin‐dependent kinase II–dependent improvements in adrenergically stimulated E‐C coupling. Deletion of β2AR abolishes sildenafil's effect. Although the PDE5‐β2AR association is not altered in HFD, phosphodiesterase 3 displays an increased association with the β2AR‐PDE5 complex in HFD myocytes.ConclusionsThis study elucidates mechanisms by which the β2AR‐PDE5 axis can be targeted for treating diabetic cardiomyopathy. Inhibition of PDE5 enhances β2AR stimulation of cGMP and cAMP signals, as well as protein kinase G–dependent E‐C coupling in HFD myocytes.