The role of connective-tissue growth factor in TGF-beta-induced anchorage-independent growth and regulation of cell cycle of fibroblasts

Connective tissue growth factor (CTGF) is a 38 kDa cysteine-rich peptide whose synthesis and secretion are uniquely induced by transforming growth factor type-beta (TGF-beta) in connective tissue cells. TGF-beta has the unique ability to stimulate the growth of normal fibroblasts in soft agar, a property of transformed cells.I have conducted experiments to investigate the role of CTGF in TGF-beta stimulated anchorage-independent growth (AIG). The results of these studies indicate that CTGF cannot substitute for TGF-beta as an inducer of AIG in NRK fibroblasts. However, CTGF is required during the induction of AIG by TGF-beta. This was confirmed by using CTGF specific antibodies and an anti-sense CTGF gene (NRK-ASCTGF), both of which inhibited TGF-beta induced AIG. It was also possible to block the TGF-beta-induction of CTGF expression and AIG by upregulating levels of intracellular cAMP through the addition of compounds such as cholera toxin (CTX), forskolin or 8-Br-cAMP to the fibroblasts. Under these conditions, AIG could be restored in a cell cycle dependent manner by addition of recombinant CTGF (rCTGF) to the cells. Neither fibroblast growth factor (FGF) nor platelet-derived growth factor (PDGF) could substitute for CTGF in this process. These studies demonstrate that the TGF-beta stimulation of NRK fibroblast AIG is dependent on events induced via the synergistic action of CTGF-dependent and CTGF-independent signaling pathways.Further detailed studies into the molecular mechanism of CTGF action indicate that CTGF controls cell cycle progression through late G1 and S-phase of NRK fibroblast suspension cultures. CTGF allows S-phase entry by upregulating cyclin A levels. The molecular mechanism for cyclin A induction appears to be via reduction of P27Kip1 levels which results in hyperphosphorylation of the retinoblastoma protein (pRb) and release of E2F, a known transcriptional regulator for the cyclin A gene. These data indicate that CTGF acts as a mediator of TGF-beta induced fibroblast proliferation in suspension cultures by modulating cyclin dependent kinase (cdk) activities

miR-221/222 compensates for Skp2-mediated p27 degradation and is a primary target of cell cycle regulation by prostacyclin and cAMP.

p27(kip1) (p27) is a cdk-inhibitory protein with an important role in the proliferation of many cell types. SCF(Skp2) is the best studied regulator of p27 levels, but Skp2-mediated p27 degradation is not essential in vivo or in vitro. The molecular pathway that compensates for loss of Skp2-mediated p27 degradation has remained elusive. Here, we combine vascular injury in the mouse with genome-wide profiling to search for regulators of p27 during cell cycling in vivo. This approach, confirmed by RT-qPCR and mechanistic analysis in primary cells, identified miR-221/222 as a compensatory regulator of p27. The expression of miR221/222 is sensitive to proteasome inhibition with MG132 suggesting a link between p27 regulation by miRs and the proteasome. We then examined the roles of miR-221/222 and Skp2 in cell cycle inhibition by prostacyclin (PGI(2)), a potent cell cycle inhibitor acting through p27. PGI(2) inhibited both Skp2 and miR221/222 expression, but epistasis, ectopic expression, and time course experiments showed that miR-221/222, rather than Skp2, was the primary target of PGI(2). PGI(2) activates Gs to increase cAMP, and increasing intracellular cAMP phenocopies the effect of PGI(2) on p27, miR-221/222, and mitogenesis. We conclude that miR-221/222 compensates for loss of Skp2-mediated p27 degradation during cell cycling, contributes to proteasome-dependent G1 phase regulation of p27, and accounts for the anti-mitogenic effect of cAMP during growth inhibition

Effect of cAMP elevating agents on miR-221/222 and p27 expression.

<p>(<b>A–B</b>) Quiescent early passage VSMCs from wild-type were stimulated with 10% FBS in the absence (control) or presence of 50 µM U0126 (U0), 1 mM 8Br-cAMP, or 100 µM Forskolin (Fsk). In A, total RNA was extracted at 24 h, and miR-221/222 expression levels were determined by RT-qPCR. Results show mean ± SE, n = 3−4. In B, total protein was extracted at 24 h and analyzed by western blotting for p27, dually phosphorylated ERK (pERK), total ERK and GAPDH (loading control). (<b>C–E</b>) The experiment in A was repeated with wild-type and p27-null VSMCs in 6-well dishes containing coverslips and EdU. In C, coverslips were fixed at 48 h and stained for EdU; results are plotted relative to the FBS-treated control; n = 3. In D-E, total RNA was extracted at 24 h, and miR-221 or miR-222 expression levels were determined by RT-qPCR. Results show mean ± SD, n = 2. (<b>F</b>) miR-221/222 regulation by mitogens, ERK, PGI₂, and cAMP.</p

miR-221/222 and Skp2 mRNA expression in injured femoral arteries.

<p>Male SMA-GFP (5–6 mo.) mice were subjected to fine-wire femoral artery injury. Injured regions of femoral arteries (as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056140#pone-0056140-g001" target="_blank">Fig. 1A</a>) and uninjured control femoral arteries were micro-dissected, and RNA was isolated. RT-qPCR was performed for miRNA221, miRNA222 and Skp2 expression. Gene expression for each mouse was expressed as fold increase (injured regions relative to uninjured control).</p

Transcript profiling reveals that miR-221/222 is induced after vascular injury in vivo.

<p>(<b>A</b>) Male SMA-GFP mice (5–6 mo) were subjected to fine-wire femoral artery injury. Injured arteries were isolated, carefully opened, and immediately imaged for GFP fluorescence. A representative image of an uninjured and injured femoral artery is shown for a single mouse. The bracket shows a region of vascular injury. (<b>B</b>) Uninjured femoral arteries and GFP-negative regions of injured femoral arteries were collected for transcript profiling. Genes differentially expressed in these tissues were plotted against the Gene Ontology (GO) category, Cellular Process. (<b>C</b>) Interaction map showing upstream regulators of p27 that are differentially expressed in injured vs. uninjured femoral arteries as determined by Ingenuity Pathway Analysis (IPA) of the microarray data. Green and red represent induction and repression, respectively. Upstream p27 regulators in the IPA database that were not differentially expressed during in vivo response to injury are uncolored. The boxed region of interest at the bottom of the interaction map is expanded below to highlight the induction of miR-221 (green oval). (<b>D</b>) Quiescent early passage mouse VSMCs were stimulated with 10% FBS for 24 h. Total RNA was collected, and the levels of miR-221/222 and Skp2 mRNA were determined by RT-qPCR. Results show mean ± SD, n = 2.</p

Effects of miR-221/222 and Skp2 on p27 levels during cell cycling and cell cycle inhibition.

<p>(<b>A</b>) Early passage VSMCs from wild-type or p27T187A mice were transiently transfected with control or anti-miR-222. Transfected cells were serum starved and stimulated with 10% FBS for 24 h before being collected and analyzed by western blotting for p27 and actin (loading control). (<b>B</b>) Cells were also serum starved and stimulated with 10% FBS for 48 h on cover slips; collected coverslips were used to determine EdU incorporation. Results show mean ± SD, n = 3.</p

miR-221/222 is a primary target of PGI₂.

<p>Quiescent early passage VSMCs from wild-type or p27-null mice were stimulated with 10% FBS in the absence (control; C) or presence of 200 nM cicaprost (cica). (<b>A</b>) Total RNA was extracted at 24 h, and Skp2 mRNA levels were determined by RT-qPCR. Results show mean ± SD, n = 2. (<b>B</b>) Total protein was extracted and analyzed by western blotting for p27, Skp2 and actin (loading control). <b>(C)</b> Total RNA was extracted at 24 h, and miR-221/222 levels were determined by RT-qPCR. Results show mean ± SD, n = 2.</p

Regulation of G1 and S phase p27 by PGI₂.

<p>(<b>A–B</b>) Quiescent early passage explant VSMCs from wild-type or p27T187A mice were stimulated with 10% FBS for 24 or 30 h in the absence or presence of 200 nM cicaprost. Total protein was extracted at the indicated times and analyzed by western blotting for p27 and actin (loading control). The percent EdU incorporation, determined from coverslips included in the experiments, is shown in italics. (<b>C–D</b>) Quiescent VSMCs were treated with 10 µM MG132 or DMSO (vehicle control) and stimulated with 10% FBS for the indicated times. Total protein was extracted and analyzed for p27 protein levels by western blotting and actin (loading control). Total RNA was extracted at 9 (n = 3) and 18 h (n = 2) and analyzed for miR221/222 by RT-qPCR. RT-qPCR results show mean ± SD. Note that G1 phase downregulation of p27 was evident 9 h after FBS stimulation in this experiment, affording us more time points to document the inhibitory effect of MG132 on G1 phase miR221/222.</p

miR221/222 regulation by PGI2 and role in mitogensis.

<p>(<b>A</b>) Serum-starved VMSCs from wild-type mice were incubated with 10% FBS and 2 nM cicaprost for selected times in the presence of EdU. Top Results show mean ± SD, n = 2. Bottom panel: S phase entry was assessed and plotted as percent maximal EdU incorporation. Results show mean ± SD, n = 3. (<b>B–C</b>) Early passage wild-type VSMCs were transiently transfected with an expression plasmid for pCDNA (control) or miRNA-222. The cells were serum starved and stimulated with 10% FBS for 24 (panel B) or 48 (panel C) h in the absence or presence of 200 nM cicaprost (cica) before being collected and analyzed for p27 levels by western blotting or S phase entry by EdU incorporation. Results in panel C show mean ± SD, n = 3.</p

Cardiovascular Protection by ApoE and ApoE-HDL Linked to Suppression of ECM Gene Expression and Arterial Stiffening

Arterial stiffening is a risk factor for cardiovascular disease, but how arteries stay supple is unknown. Here, we show that apolipoprotein E (apoE) and apoE-containing high-density lipoprotein (apoE-HDL) maintain arterial elasticity by suppressing the expression of extracellular matrix genes. ApoE interrupts a mechanically driven feed-forward loop that increases the expression of collagen-I, fibronectin, and lysyl oxidase in response to substratum stiffening. These effects are independent of the apoE lipid-binding domain and transduced by Cox2 and miR-145. Arterial stiffness is increased in apoE null mice. This stiffening can be reduced by administration of the lysyl oxidase inhibitor BAPN, and BAPN treatment attenuates atherosclerosis despite highly elevated cholesterol. Macrophage abundance in lesions is reduced by BAPN in vivo, and monocyte/macrophage adhesion is reduced by substratum softening in vitro. We conclude that apoE and apoE-containing HDL promote healthy arterial biomechanics and that this confers protection from cardiovascular disease independent of the established apoE-HDL effect on cholesterol