Hyaluronan and CD44 antagonize mitogen-dependent cyclin D1 expression in mesenchymal cells

High molecular weight (HMW) hyaluronan (HA) is widely distributed in the extracellular matrix, but its biological activities remain incompletely understood. We previously reported that HMW-HA binding to CD44 antagonizes mitogen-induced S-phase entry in vascular smooth muscle cells (SMCs; Cuff, C.A., D. Kothapalli, I. Azonobi, S. Chun, Y. Zhang, R. Belkin, C. Yeh, A. Secreto, R.K. Assoian, D.J. Rader, and E. Puré. 2001. J. Clin. Invest. 108:1031–1040); we now characterize the underlying molecular mechanism and document its relevance in vivo. HMW-HA inhibits the mitogen-dependent induction of cyclin D1 and down-regulation of p27kip1 in vascular SMCs. p27kip1 messenger RNA levels were unaffected by HMW-HA, but the expression of Skp2, the rate-limiting component of the SCF complex that degrades p27kip1, was reduced. Rescue experiments identified cyclin D1 as the primary target of HMW-HA. Similar results were observed in fibroblasts, and these antimitogenic effects were not detected in CD44-null cells. Analysis of arteries from wild-type and CD44-null mice showed that the effects of HMW-HA/CD44 on cyclin D1 and Skp2 gene expression are detected in vivo and are associated with altered SMC proliferation after vascular injury

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