Rommel L. Sagana

CONTROL OF FIBROBLAST FIBRONECTIN EXPRESSION AND ALTERNATIVE SPLICING VIA THE PI3K/AKT/mTOR PATHWAY

Fibronectin (FN), a ubiquitous glycoprotein that plays critical roles in physiologic and pathologic conditions, undergoes alternative splicing which distinguishes plasma FN (pFN) from cellular FN (cFN). Although both pFN and cFN can be incorporated into the extracellular matrix, a distinguishing feature of cFN is the inclusion of an alternatively spliced exon termed EDA (for extra type III domain A). The molecular steps involved in EDA splicing are well-characterized, but pathways influencing EDA splicing are less clear. We have previously found an obligate role for inhibition of the tumor suppressor phosphatase and tensin homologue on chromosome 10 (PTEN), the primary regulator of the PI3K/Akt pathway, in fibroblast activation. Here we show TGF-beta, a potent inducer of both EDA splicing and fibroblast activation, inhibits PTEN expression and activity in mesenchymal cells, corresponding with enhanced PI3K/Akt signaling. In pten(-/-) fibroblasts, which resemble activated fibroblasts, inhibition of Akt attenuated FN production and decreased EDA alternative splicing. Moreover, inhibition of mammalian target of rapamycin (mTOR) in pten(-/-) cells also blocked FN production and EDA splicing. This effect was due to inhibition of Akt-mediated phosphorylation of the primary EDA splicing regulatory protein SF2/ASF. Importantly, FN silencing in pten(-/-) cells resulted in attenuated proliferation and migration. Thus, our results demonstrate that the PI3K/Akt/mTOR axis is instrumental in FN transcription and alternative splicing, which regulates cell behavior.

X-linked inhibitor of apoptosis regulates lung fibroblast resistance to Fas-mediated apoptosis.

The accumulation of apoptosis-resistant fibroblasts within fibroblastic foci is a characteristic feature of idiopathic pulmonary fibrosis (IPF), but the mechanisms underlying apoptosis resistance remain unclear. A role for the inhibitor of apoptosis (IAP) protein family member X-linked inhibitor of apoptosis (XIAP) has been suggested by prior studies showing that (1) XIAP is localized to fibroblastic foci in IPF tissue and (2) prostaglandin E₂ suppresses XIAP expression while increasing fibroblast susceptibility to apoptosis. Based on these observations, we hypothesized that XIAP would be regulated by the profibrotic mediators transforming growth factor (TGF)β-1 and endothelin (ET)-1 and that increased XIAP would contribute to apoptosis resistance in IPF fibroblasts. To address these hypotheses, we examined XIAP expression in normal and IPF fibroblasts at baseline and in normal fibroblasts after treatment with TGF-β1 or ET-1. The role of XIAP in the regulation of fibroblast susceptibility to Fas-mediated apoptosis was examined using functional XIAP antagonists and siRNA silencing. In concordance with prior reports, fibroblasts from IPF lung tissue had increased resistance to apoptosis compared with normal lung fibroblasts. Compared with normal fibroblasts, IPF fibroblasts had significantly but heterogeneously increased basal XIAP expression. Additionally, TGF-β1 and ET-1 induced XIAP protein expression in normal fibroblasts. Inhibition or silencing of XIAP enhanced the sensitivity of lung fibroblasts to Fas-mediated apoptosis without causing apoptosis in the absence of Fas activation. Collectively, these findings support a mechanistic role for XIAP in the apoptosis-resistant phenotype of IPF fibroblasts.

Achieving Zero Central Line–associated Bloodstream Infection Rates in Your Intensive Care Unit

Central line-associated bloodstream infection (CLABSI) is one of the most common health care-associated infections in the United States. The costs associated with CLABSIs include an estimated 28,000 deaths in the intensive care unit and up to

Phosphatase and tensin homologue on chromosome 10 (PTEN) directs prostaglandin E2-mediated fibroblast responses via regulation of E prostanoid 2 receptor expression.

2.3 billion annually. Best practice guidelines, checklists, and establishing a culture of safety in hospitals are all initiatives designed to reduce the rate of CLABSI to zero.

Management of sepsis and septic shock

Prostaglandin E2 (PGE2) is an arachidonic acid metabolite that counters transforming growth factor-β-induced fibroblast activation via E prostanoid 2 (EP2) receptor binding. Phosphatase and tensin homologue on chromosome 10 (PTEN) is a lipid phosphatase that, by antagonizing the phosphoinositol 3-kinase (PI3K) pathway, also inhibits fibroblast activation. Here, we show that PTEN directly regulates PGE2 inhibition of fibroblast activation by augmenting EP2 receptor expression. The increase in collagen production and α-smooth muscle actin expression observed in fibroblasts in which PTEN is deficient was resistant to the usual suppressive effects of PGE2. This was due to marked down-regulation of EP2, a Gs protein-coupled receptor (GPCR) that mediates the inhibitory actions of this prostanoid via cAMP. pten−/− or PTEN-inhibited fibroblasts in which the PI3K pathway was blocked demonstrated a restoration of EP2 receptor expression, due to augmented gene transcription and mRNA instability. Importantly, restoration of the balance between PI3K and PTEN reestablished the inhibitory effect of PGE2 on fibroblast activation. No such influence of PTEN was observed on alternative E prostanoid GPCRs. Moreover, our studies identified a positive feedback loop in which cAMP signaling enhanced EP2 receptor expression, independent of PTEN. Therefore, our findings indicate that PTEN regulates the antifibrotic effects of PGE2 by a specific and permissive effect on EP2 receptor expression. Further, our data imply that cAMP signaling circumvents EP2 down-regulation in pten-deficient cells to restore EP2 receptor expression. This is the first description, to our knowledge, of PI3K/PTEN balance directing GPCR expression, and provides a novel mechanism for cellular effects of PTEN.

Increased survivin expression contributes to apoptosis-resistance in IPF fibroblasts

Despite advances in modern medicine, sepsis and septic shock continue to be a leading cause of death worldwide. The United States alone has over 750,000 reported cases per year. It is estimated that severe sepsis is diagnosed in 2 % of all hospital admissions resulting in 10 % of all ICU admissions. The diagnosis and management of sepsis have greatly evolved with the creation of care “bundles” such as Early Goal Directed Therapy incorporated into the Surviving Sepsis Campaign. Research results continue to refine the proposed guidelines in the treatment of this disease.