Jill Kleidon

Pathologic Caveolin-1 Regulation of PTEN in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive fibroproliferative disorder refractory to current pharmacological therapies. Fibroblasts isolated from IPF patients display pathological activation of PI3K/Akt caused by low PTEN phosphatase activity. This enables these cells to escape the negative proliferative properties of polymerized collagen. The mechanism underlying low PTEN activity in IPF fibroblasts is unclear, but our prior studies indicate that membrane-associated PTEN expression is decreased in these cells. Caveolin-1 is an integral membrane protein whose expression is decreased in IPF lung tissue, but how low caveolin-1 contributes to pathological fibrosis is incompletely understood. The objective of this study was to examine the hypothesis that caveolin-1 regulates PTEN function in IPF fibroblasts. Here we demonstrate that caveolin-1 expression is a determinant of membrane PTEN levels and show that PTEN interacts with caveolin-1 via its caveolin-1-binding sequence. We demonstrate that caveolin-1 expression is low in IPF fibroblasts and that this correlates with low membrane PTEN levels, whereas overexpression of caveolin-1 restores membrane PTEN levels, inhibits Akt phosphorylation, and suppresses proliferation. We demonstrate that caveolin-1 and PTEN expression are low in myofibroblasts within IPF fibroblastic foci. These data indicate that IPF fibroblasts display low caveolin-1 expression, which results in low membrane-associated PTEN expression. This creates a membrane microenvironment depleted of inhibitory phosphatase activity, facilitating the aberrant activation PI3K/Akt and pathological proliferation.

PTEN Regulates Fibroblast Elimination during Collagen Matrix Contraction

During tissue repair, excess fibroblasts are eliminated by apoptosis. This physiologic process limits fibrosis and restores normal anatomic patterns. Replicating physiologic apoptosis associated with tissue repair, fibroblasts incorporated into type I collagen matrices undergo apoptosis in response to collagen matrix contraction. In this in vitro model of wound repair, fibroblasts first attach to collagen via α2β1 integrin. This provides a survival signal via activation of the phosphatidylinositol 3-kinase/Akt signal pathway. However, during subsequent collagen matrix contraction, the level of phosphorylated Akt progressively declines, triggering apoptosis. The mechanism underlying the fall in phosphorylated Akt is incompletely understood. Here we show that PTEN phosphatase becomes activated during collagen matrix contraction and is responsible for antagonizing phosphatidylinositol 3-kinase activity and promoting a decline in phosphorylated Akt and fibroblast apoptosis in response to collagen contraction. PTEN null fibroblasts displayed enhanced levels of phosphorylated Akt and were resistant to collagen matrix contraction-induced apoptosis. Reconstitution of PTEN in PTEN null cells conferred susceptibility to apoptosis in response to contraction of collagen matrices. Consistent with this, knockdown of PTEN in PTEN+/+ embryonic fibroblasts by small interfering RNA augmented Akt activity and suppressed apoptosis in contractile collagen matrices. Furthermore, inhibition of Akt activity restored the sensitivity of PTEN null cells to collagen contraction-induced apoptosis, indicating that the mechanism by which PTEN alters fibroblast viability is through modulation of phosphorylated Akt levels. Our work suggests that collagen matrix contraction activates PTEN by a mechanism involving cytoskeletal disassembly. Our studies indicate a key role for PTEN in regulating fibroblast viability during tissue repair.

Polymerized Collagen Inhibits Fibroblast Proliferation via a Mechanism Involving the Formation of a β1 Integrin-Protein Phosphatase 2A-Tuberous Sclerosis Complex 2 Complex That Suppresses S6K1 Activity

Polymerized type I collagen suppresses fibroblast proliferation. Previous studies have implicated inhibition of fibroblast proliferation with polymerized collagen-mediated suppression of S6K1, but the molecular mechanism of the critical negative feedback loop has not yet been fully elucidated. Here, we demonstrate that polymerized collagen suppresses G1/S phase transition and fibroblast proliferation by a novel mechanism involving the formation of a β1 integrin-protein phosphatase 2A (PP2A)-tuberous sclerosis complex 2 (TSC2) complex that represses S6K1 activity. In response to fibroblast interaction with polymerized collagen, β1 integrin forms a complex with PP2A that targets TSC2 as a substrate. PP2A represses the level of TSC2 phosphorylation and maintains TSC2 in an activated state. Activated TSC2 negatively regulates the downstream kinase S6K1 and inhibits G1/S transit. Knockdown of TSC2 enables fibroblasts to overcome the anti-proliferative properties of polymerized collagen. Furthermore, we show that this reduction in TSC2 and S6K1 phosphorylation occurs largely independent of Akt. Although S6K1 activity was markedly suppressed by polymerized collagen, we found that minimal changes in Akt activity occurred. We demonstrate that up-regulation of Akt by overexpression of constitutively active phosphatidylinositol 3-kinase p110 subunit had minor effects on TSC2 and S6K1 phosphorylation. These findings demonstrate that polymerized collagen represses fibroblast proliferation by a mechanism involving the formation of a β1 integrin-PP2A-TSC2 complex that negatively regulates S6K1 and inhibits G1/S phase transition.

An HSV-TK Transgenic Mouse Model to Evaluate Elimination of Fibroblasts for Fibrosis Therapy

Pathological fibroproliferation after tissue injury is harmful and may lead to organ dysfunction. Unfortunately, fibroproliferative diseases remain intractable to current therapeutic strategies. Thus, new therapeutic approaches are needed. One possible approach is to promote resolution of physiological fibroproliferation that follows injury before it becomes pathological by activating apoptosis selectively in fibrotic lesions. However, it is not known whether selective elimination of fibroblasts will prevent fibrosis or impede repair or worsen injury by eliminating topographic signals essential to organ reconstitution. To address this question, a tractable in vivo model system is needed in which fibroblasts can be targeted to undergo apoptosis at a chosen time and place. We developed transgenic mice expressing HSV-TK from the type I collagen promoter to determine whether selective elimination of fibroblasts actively forming fibrotic lesions is an effective therapeutic strategy for fibroproliferative disorders. The transgene renders fibroblasts actively forming fibrotic tissue susceptible to ganciclovir. To validate the transgenic model we examined whether administration of ganciclovir prevents the development of fibrosis in sponges implanted subcutaneously in the backs of the transgenic mice. We demonstrate that fibroblasts/myofibroblasts isolated from sponges express HSV-TK protein and are selectively ablated by ganciclovir in vitro. In adult transgenic mice, ganciclovir treatment attenuated the development of fibrotic tissue in the sponges both biochemically and histologically. We conclude that this transgenic model system is an ideal approach to determine whether targeted ablation of fibroblasts is an effective therapeutic strategy for fibrotic diseases.

Pathological integrin signaling enhances proliferation of primary lung fibroblasts from patients with idiopathic pulmonary fibrosis

Xia et al. 2008. J. Exp. Med. doi:10.1084/jem.20080001 [OpenUrl][1][Abstract/FREE Full Text][2] [1]: {openurl}?query=rft_id%253Dinfo%253Adoi%252F10.1084%252Fjem.20080001%26rft_id%253Dinfo%253Apmid%252F18541712%26rft.genre%253Darticle%26rft_val_fmt%253Dinfo%253Aofi%252Ffmt%253Akev%253Amtx%