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The proinflammatory cytokine IFN-γ is a master regulator of atherosclerosis and mediates its cellular actions mainly through STAT1. Unfortunately, the impact of other IFN-γ inducible pathways on STAT1 activation and the regulation of downstream responses associated with atherosclerosis in human macrophages are poorly understood and were therefore investigated. In this study, we demonstrate that the IFN-γ–mediated phosphorylation of STAT1 on Ser727, crucial for its maximal activity, was attenuated in human macrophages by pharmacological inhibition of ERK. In these cells, IFN-γ induced changes in the expression of several key genes implicated in atherosclerosis, such as MCP-1, through an ERK-dependent mechanism. Additionally, the IFN-γ–induced activity of STAT1-responsive promoters was attenuated by transfection of dominant-negative forms of ERK and other key components of this pathway. Furthermore, the IFN-γ–induced uptake of acetylated and oxidized low-density lipoprotein by human macrophages was attenuated by pharmacological inhibition or RNA interference–mediated knockdown of ERK. These studies suggest a critical role for ERK signaling in the IFN-γ–mediated changes in macrophage cholesterol homeostasis and gene expression during atherosclerosis.

Aberrant expression of the human hyaluronan synthase 2 (HAS2) gene has been implicated in the pathology of malignancy, pulmonary arterial hypertension, osteoarthritis, asthma, thyroid dysfunction, and large organ fibrosis. Renal fibrosis is associated with increased cortical synthesis of hyaluronan (HA), an extracellular matrix glycosaminoglycan, and we have shown that HA is a correlate of interstitial fibrosis in vivo. Our previous in vitro data have suggested that both HAS2 transcriptional induction and subsequent HAS2-driven HA synthesis may contribute to kidney fibrosis via phenotypic modulation of the renal proximal tubular epithelial cell (PTC). Post-transcriptional regulation of HAS2 mRNA synthesis by the natural antisense RNA HAS2-AS1 has recently been described in osteosarcoma cells, but the antisense transcript was not detected in kidney. In this study, PTC stimulation with IL-1␤ or TGF-␤1 induced coordinated temporal profiles of HAS2-AS1 and HAS2 transcription. Constitutive activity of the putative HAS2-AS1 promoter was demonstrated, and transcription factor-binding sequence motifs were identified. Knockdown of Sp1/Sp3 expression by siRNA blunted IL-1␤ induction of both HAS2-AS1 and HAS2, and Smad2/Smad3 knockdown similarly attenuated TGF-␤1 stimulation. Inhibition of IL-1␤-stimulated HAS2-AS1 RNA induction using HAS2-AS1-specific siRNAs also suppressed upregulation of HAS2 mRNA transcription. The thermodynamic feasibility of HAS2-AS1/HAS2 heterodimer formation was demonstrated in silico, and locus-specific cytoplasmic doublestranded RNA was detected in vitro. In summary, our data show that transcriptional induction of HAS2-AS1 and HAS2 occurs simultaneously in PTCs and suggest that transcription of the antisense RNA stabilizes or augments HAS2 mRNA expression in these cells via RNA/mRNA heteroduplex formation.The linear non-sulfated glycosaminoglycan hyaluronan (HA) 5 is a ubiquitous component of the vertebrate extracellular matrix with a multiplicity of cellular functions in both physiological and pathophysiological contexts (1-8). HA is synthesized at the cell membrane by the HA synthase (HAS) enzymes, encoded in humans by the corresponding multigene family HAS1-3 (9 -11).Frequently associated with the fibrotic response in major organs, HA is a highly effective biomarker for liver fibrosis (12,13). Similarly, in lung fibrosis, accumulation of HA has been observed (14, 15), and in some cases, up-regulated HAS2 transcription has also been reported (14). For all progressive renal diseases, the rate of progression correlates with the degree of corticointerstitial fibrosis. Increased HA synthesis and extracellular matrix expansion in the renal corticointerstitium are common features of kidney fibrosis (16), and our recent studies on renal biopsy samples from diabetic nephropathy patients demonstrate that HA is a correlate of interstitial fibrosis in vivo (17). A number of our in vitro studies have suggested that HA may contribute to renal fibrosis via regulation of the renal proximal tubular epithelia...

The anti-atherogenic cytokine, TGF-β, plays a key role during macrophage foam cell formation by modulating the expression of key genes involved in the control of cholesterol homeostasis. Unfortunately, the molecular mechanisms underlying these actions of TGF-β remain poorly understood. In this study we examine the effect of TGF-β on macrophage cholesterol homeostasis and delineate the role of Smads-2 and ‐3 during this process. Western blot analysis showed that TGF-β induces a rapid phosphorylation-dependent activation of Smad-2 and ‐3 in THP-1 and primary human monocyte-derived macrophages. Small interfering RNA-mediated knockdown of Smad-2/3 expression showed that the TGF-β-mediated regulation of key genes implicated in the uptake of modified low density lipoproteins and the efflux of cholesterol from foam cells was Smad-dependent. Additionally, through the use of virally delivered Smad-2 and/or Smad-3 short hairpin RNA, we demonstrate that TGF-β inhibits the uptake of modified LDL by macrophages through a Smad-dependent mechanism and that the TGF-β-mediated regulation of CD36, lipoprotein lipase and scavenger receptor-A gene expression was dependent on Smad-2. These studies reveal a crucial role for Smad signaling, particularly Smad-2, in the inhibition of foam cell formation by TGF-β through the regulation of expression of key genes involved in the control of macrophage cholesterol homeostasis.

Liver X receptors (LXRs) belong to the nuclear receptor superfamily of ligand-dependent transcription factors. LXRs are activated by oxysterols, metabolites of cholesterol, and therefore act as intracellular sensors of this lipid. There are two LXR genes (α and β) that display distinct tissue/cell expression profiles. LXRs interact with regulatory sequences in target genes as heterodimers with retinoid X receptor. Such direct targets of LXR actions include important genes implicated in the control of lipid homeostasis, particularly reverse cholesterol transport. In addition, LXRs attenuate the transcription of genes associated with the inflammatory response indirectly by transrepression. In this review, we describe recent evidence that both highlights the key roles of LXRs in atherosclerosis and inflammation and provides novel insights into the mechanisms underlying their actions. In addition, we discuss the major limitations of LXRs as therapeutic targets for the treatment of atherosclerosis and how these are being addressed.

HighlightsMacrophages can internalise LDL through scavenger receptor-independent mechanisms.Macropinocytosis has been shown to contribute significantly to foam cell formation.Cytokines such as TGF-β, IL-33, IFN-γ and IL-17A can modulate macropinocytosis.TGF-β mediated inhibition of macropinocytosis is a Smad-2/-3-independent process.Macropinocytosis is a promising target for therapeutic intervention of atherosclerosis.

The linear glycosaminoglycan hyaluronan (HA) is synthesized at the plasma membrane by the HA synthase (HAS) enzymes HAS1, -2, and -3 and performs multiple functions as part of the vertebrate extracellular matrix. Up-regulation of HA synthesis in the renal corticointerstitium, and the resultant extracellular matrix expansion, is a common feature of renal fibrosis. However, the regulation of expression of these HAS isoforms at transcriptional and translational levels is poorly understood. We have recently described the genomic structures of the human HAS genes, thereby identifying putative promoter regions for each isoform. Further analysis of the HAS2 gene identified the transcription initiation site and showed that region F3, comprising the proximal 121 bp of promoter sequence, mediated full constitutive transcription. In the present study, we have analyzed this region in the human renal proximal tubular epithelial cell line HK-2. Electrophoretic mobility shift and promoter assay data demonstrated that transcription factors Sp1 and Sp3 bound to three sites immediately upstream of the HAS2 transcription initiation site and that mutation of the consensus recognition sequences within these sites ablated their transcriptional response. Furthermore, subsequent knockdown of Sp1 or Sp3 using small interfering RNAs decreased constitutive HAS2 mRNA synthesis. In contrast, significant binding of HK-2 nuclear proteins by putative upstream NF-Y, CCAAT, and NF-B recognition sites was not observed. The identification of Sp1 and Sp3 as principal mediators of HAS2 constitutive transcription augments recent findings identifying upstream promoter elements and provides further insights into the mechanism of HAS2 transcriptional activation. Hyaluronan (HA)3 is a linear non-sulfated glycosaminoglycan found commonly in the vertebrate extracellular matrix and which has a variety of functions during and following development (1-6). HA is synthesized by the HA synthase (HAS) enzymes that are encoded by the corresponding multigene family HAS1, -2, -3a, and -3b (7-10), and its importance in the extracellular matrix is underlined by the expanding range of pathological contexts in which modified or aberrant HA metabolism appears to play a role. These include malignancy, osteoarthritis, and pulmonary and vascular disorders, along with other immune and inflammatory diseases (11)(12)(13)(14)(15)(16)(17)(18)(19). HA has also been implicated in regenerative processes such as wound healing (e.g. and as a key mediator of the immune process (19).Under homeostasis in the healthy kidney, the expression of HA in the cortical interstitium is low, with high levels found only in the renal papilla. Following acute ischemic injury, interstitial inflammation, or during progressive renal fibrosis, however, there is greatly increased peritubular expression of both HA and the cell surface HA receptor CD44 in the cortex (23)(24)(25)(26)(27)(28). This suggests that alterations in HA synthesis and turnover may be involved either in the maintenance of homeostasis...