The Src-activating and signaling molecule (Srcasm) is a recently described activator and substrate of Src-family tyrosine kinases (SFKs). When phosphorylated at specific tyrosines, Srcasm associates with Grb2 and p85, the regulatory subunit of phosphoinositide 3-kinase; however, little is known about the role of Srcasm in cellular signaling. Data presented here demonstrate that epidermal growth factor (EGF) receptor ligands promote the tyrosine phosphorylation of endogenous and adenovirally transduced Srcasm in keratinocytes, and that increased levels of Srcasm activate endogenous SFKs, with a preference for Fyn and Src. In addition, Srcasm potentiates EGF-dependent signals transmitted by SFKs in keratinocytes. Tyrosine phosphorylation of Srcasm is dependent on growth factors and the activity of EGFR and SFKs. Increased Srcasm expression enhances p44/42 mitogen-activated protein kinase activity and Elk-1-dependent transcriptional events. Elevated Srcasm levels inhibit keratinocyte proliferation while promoting specific aspects of keratinocyte differentiation. Lastly, Srcasm levels are decreased in human cutaneous neoplasia. Collectively, these data demonstrate that Srcasm plays a role in linking EGF receptor- and SFK-dependent signaling to differentiation in keratinocytes.

Src family tyrosine kinases (SFK) regulate cell proliferation, and increased SFK activity is common in human carcinomas, including cutaneous squamous cell carcinomas (SCC) and its precursors. The elevated SFK activity in cutaneous SCCs was modeled using K14-Fyn Y528F transgenic mice, which spontaneously form punctate keratotic lesions, scaly plaques, and large tumors resembling actinic keratoses, SCC in situ, and SCCs, respectively. Lesional tissue showed increased levels of activated SFKs, PDK1, STAT3, and ERK1/2, whereas Notch1/ NICD protein and transcript levels were decreased. p53 levels also were decreased in SCC in situ and SCCs. Increasing Srcasm levels using a K14-Fyn Y528F/K14-Srcasm double transgenic model markedly inhibited cutaneous neoplasia.

Background The bioactive steroid, marinobufagenin (MBG), is an endogenous Na/K-ATPase bufadienolide inhibitor that is synthetized by adrenocortical and placental cells. MBG binding to Na/K-ATPase initiates pro-fibrotic cell signaling, and heightened MBG levels are implicated in the pathogenesis of hypertension, preeclampsia and chronic kidney disease. Steroids are derived from cholesterol through the “traditional” steroidogenesis pathway initiated by enzyme CYP11A1, and via the “acidic” bile acid pathway, which is controlled by enzyme CYP27A1. The mechanism of MBG biosynthesis in mammals however remains unknown. Methods and Results Here we show that post-transcriptional silencing of the CYP27A1 gene in human trophoblast and rat adrenocortical cells reduced the expression of CYP27A1 mRNA by 70%, reduced total bile acids 2-fold, and MBG levels by 67%, compared to non-treated cells or cells transfected with non-targeting siRNA. In contrast, silencing of the CYP11A1 gene did not affect MBG production in either cell culture, but suppressed production of progesterone 2-fold in human trophoblast cells, and of corticosterone by 90% in rat adrenocortical cells, compared to cells transfected with non-targeting siRNA. In vivo, in a high salt administration experiment, male and female Dahl-S rats became hypertensive after 4 weeks on a high NaCl diet, their plasma MBG levels doubled, and adrenocortical CYP27A1 mRNA and protein increased 1.6-fold and 2.0-fold. Conclusions Therefore, the endogenous steroidal Na/K-ATPase inhibitor, MBG, is synthesized in mammalian placenta and adrenal cortex from cholesterol through the novel “acidic” bile acid pathway. These findings will help to understand the role of MBG in highly prevalent human cardiovascular diseases.

Objective Endogenous cardiotonic steroids (CTS), including marinobufagenin (MBG), stimulate vascular synthesis of collagen. Because mineralocorticoid antagonists competitively antagonize effect of CTS on the Na/K-ATPase, we hypothesized that spironolactone would reverse the pro-fibrotic effects of MBG. Methods Experiment 1. Explants of thoracic aortae and aortic vascular smooth muscle cells (VSMC) from Wistar rats were cultured for 24 hours in the presence of vehicle or MBG (100 nmol/L) with or without canrenone (10 µmol/L), an active metabolite of spironolactone. Experiment 2. In 16 patients (56 ± 2 yrs) with resistant hypertension (RH) on a combined (Lisinopril / amlodipine / hydrochlorothiazide) therapy, we determined arterial pressure, pulse wave velocity (PWV), plasma MBG, and erythrocyte Na/K-ATPase before and six months after addition of placebo (n=8) or spironolactone (50 mg/day; n=8) to the therapy. Results In rat aortic explants and in VSMC, pretreatment with MBG resulted in a two-fold rise in collagen-1, and a marked reduction in the sensitivity of the aortic rings to the vasorelaxant effect of sodium nitroprusside following endothelin-1-induced constriction (EC50=480±67 nmol/L vs. 23±3 nmol/L in vehicle-treated rings; P<0.01). Canrenone blocked effects of MBG on collagen synthesis and restored sensitivity of vascular rings to sodium nitroprusside (EC50 = 17±1 nmol/L). RH patients exhibited elevated plasma MBG (0.42 ± 0.07 vs. 0.24 ± 0.03 nmol/L; P=0.01) and reduced Na/K-ATPase activity (1.9 ± 0.15 vs 2.8 ± 0.2 µmol Pi/ml/hr, P<0.01) vs. 7 healthy subjects. Six-month administration of spironolactone, unlike placebo treatment, was associated with a decrease in PWV and arterial pressure, and with restoration of Na/K-ATPase activity in the presence of unchanged MBG levels. Conclusion MBG-induced vascular fibrosis is a likely target for spironolactone.