Differential activation of NF-κB and AP-1 in increased fibronectin synthesis in target organs of diabetic complications

Abstract

Increased extracellular matrix protein production leading to structural abnormalities is a characteristic feature of chronic diabetic complications. We previously showed that high glucose in endothelial cell culture leads to the upregulation of basement membrane protein fibronectin (FN) via an endothelin (ET)-dependent pathway involving activation of NF-κB and activating protein-1 (AP-1). To delineate the mechanisms of basement membrane thickening, we used an animal model of chronic diabetes and evaluated ET-dependent activation of NF-κB and AP-1 and subsequent upregulation of FN in three target organs of chronic diabetic complications. After 3 mo of diabetes, retina, renal cortex, and myocardium demonstrated increased FN mRNA and increased ET-1 mRNA expression. Increased FN expression was shown to be dependent on ET receptor-mediated signaling, as the increase was prevented by the dual ET receptor antagonist bosentan. NF-κB activation was most pronounced in the retina, followed by kidney and heart. AP-1 activation was also most pronounced in the retina but was similar in both kidney and heart. Bosentan treatment prevented NF-κB activation in the retina and heart and AP-1 activation in the retina and kidney. These data indicate that, although ETs are important in increased FN production due to diabetes, the mechanisms with respect to transcription factor activation may vary depending on the microenvironment of the organ.

Extracellular signal-regulated kinase (ERK) in glucose-induced and endothelin-mediated fibronectin synthesis

“…[1][2][3][4][5]18 We have previously demonstrated an important role of hyperglycemia-induced ET-1 expression in increased ECM protein deposition. 4,15,18 We have also shown that hyperhexosemia-induced increased FN mRNA expression can be prevented by bosentan treatment in retinas and kidneys of both diabetic and galactose-fed rats. 4,15 Bosentan treatment also prevented retinal capillary and glomerular basement membrane thickening in these animals.…”

“…We have previously demonstrated that both NF-kB and AP-1 may be of importance in this scenario. 6,18 EMSA showed glucose-induced activation of both NF-kB and AP-1 in endothelial cells ( Figure 7). Inhibition of MAPK pathway caused marked attenuation of both NF-kB and AP-1 activation (Figure 7).…”

“…PCR reactions were performed essentially as described. 18 The reaction mixture consisted of 10 ml of ReadyMix SYBRt (SigmaAldrich, Canada), 1.6 ml of 25 mM MgCl 2, 1 ml of each forward and reverse 10 mM primers (Table 1), 5.4 ml H 2 O, and 1 ml complementary DNA (cDNA) template. Messenger RNA (mRNA) levels were quantified using the standard curve method.…”

Potential Contributory Role of H-Ras, a Small G-Protein, in the Development of Retinopathy in Diabetic Rats

“…Total RNA was extracted using Trizol (Invitrogen), the samples were centrifuged (12,000g ϫ 15 min), and RNA was isolated from the aqueous layer and precipitated using isopropyl alcohol. RNA (3 g) was used for cDNA synthesis according to the methods described previously (30). Briefly, RNA was mixed with oligo-d(T) primers and incubated for 10 min at 70°C.…”

Recent novel approaches to limit oxidative stress and inflammation in diabetic complications

“…Once activated, NF‐κB, additionally drives the expression of numerous growth factors, such as connective tissue growth factor (CTGF) and TGF‐β 113. This, in turn, drives the production of extracellular matrix (ECM) proteins in the heart, including collagen, fibronectin and proteoglycans, leading to fibrosis, LV remodelling and stiffness, and reduced relaxation of the heart 108, 117. Moreover, a clinical study showed a correlation between macrophage inhibitory factor and the development of diastolic dysfunction in diabetic patients 116.…”