Transforming growth factor-beta receptor signalling is modulated by integrin-linked kinase

Transforming growth factor-beta 1 (TGF-β1) modulates regeneration after injury through induction of fibroblast proliferation, migration, and differentiation into myofibroblasts. Induction of myofibroblast differentiation by TGF-β1 requires expression of integrin-linked kinase (ILK). I now show that ILK interacts with TGF-β receptor type II (TβRII) in primary dermal fibroblasts. Further, colocalization of ILK and TβRII can be observed at the cell membrane and in intracellular vesicles. The association of TβRII and ILK does not require TGF-β1 stimulation, kinase activity of TGF-β1 receptor type I or TβRII, and it does not involve interactions between ILK and focal adhesion-associated proteins. When this interaction is abolished by targeted inactivation of the Ilk gene, TβR signalling is diminished, as demonstrated by decreased phosphorylated SMAD2 levels in response to TGF-β1 treatment. This can be restored by exogenous expression of human ILK or by inhibition of TβRII degradatory pathway. Thus, ILK is essential for normal TβR signalling

Modulation of type II TGF-β receptor degradation by integrin-linked kinase

Cutaneous responses to injury, infection, and tumor formation involve the activation of resident dermal fibroblasts and subsequent transition to myofibroblasts. The key for induction of myofibroblast differentiation is the activation of transforming growth factor-β (TGF-β) receptors and stimulation of integrins and their associated proteins, including integrin-linked kinase (ILK). Cross-talk processes between TGF-β and ILK are crucial for myofibroblast formation, as ILK-deficient dermal fibroblasts exhibit impaired responses to TGF-β receptor stimulation. We now show that ILK associates with type II TGF-β receptors (TβRII) in ligand- and receptor kinase activity-independent manners. In cells with targeted Ilk gene inactivation, cellular levels of TβRII are decreased, through mechanisms that involve enhanced ubiquitination and proteasomal degradation. Partitioning of TGF-β receptors into membrane has been linked to proteasome-dependent receptor degradation. We found that interfering with membrane raft formation in ILK-deficient cells restored TβRII levels and signaling. These observations support a model whereby ILK functions in fibroblasts to direct TβRII away from degradative pathways during their differentiation into myofibroblasts

Temporal and Molecular Analyses of Cardiac Extracellular Matrix Remodeling following Pressure Overload in Adiponectin Deficient Mice.

Adiponectin, circulating levels of which are reduced in obesity and diabetes, mediates cardiac extracellular matrix (ECM) remodeling in response to pressure overload (PO). Here, we performed a detailed temporal analysis of progressive cardiac ECM remodelling in adiponectin knockout (AdKO) and wild-type (WT) mice at 3 days and 1, 2, 3 and 4 weeks following the induction of mild PO via minimally invasive transverse aortic banding. We first observed that myocardial adiponectin gene expression was reduced after 4 weeks of PO, whereas increased adiponectin levels were detected in cardiac homogenates at this time despite decreased circulating levels of adiponectin. Scanning electron microscopy and Masson's trichrome staining showed collagen accumulation increased in response to 2 and 4 weeks of PO in WT mice, while fibrosis in AdKO mice was notably absent after 2 weeks but highly apparent after 4 weeks of PO. Time and intensity of fibroblast appearance after PO was not significantly different between AdKO and WT animals. Gene array analysis indicated that MMP2, TIMP2, collagen 1α1 and collagen 1α3 were induced after 2 weeks of PO in WT but not AdKO mice. After 4 weeks MMP8 was induced in both genotypes, MMP9 only in WT mice and MMP1α only in AdKO mice. Direct stimulation of primary cardiac fibroblasts with adiponectin induced a transient increase in total collagen detected by picrosirius red staining and collagen III levels synthesis, as well as enhanced MMP2 activity detected via gelatin zymography. Adiponectin also enhanced fibroblast migration and attenuated angiotensin-II induced differentiation to a myofibroblast phenotype. In conclusion, these data indicate that increased myocardial bioavailability of adiponectin mediates ECM remodeling following PO and that adiponectin deficiency delays these effects

Regulation of ECM-related gene expressions following PO.

<p>Analysis of myocardial collagen, MMP, and TIMP expression from Ad KO or wt C57BL/6 mice 2 or 4 weeks following MTAB surgery. (A&B) Quantification of PO fold over sham changes in myocardial mRNA expressions represented as average C(t) value fold sham, where sham is set to 1. (C) Graphical representation of fold over sham mRNA change of collagens (up: blue, down: brown), MMPs (up: green, down: purple), and TIMPs (up: teal, down: orange). (C&D) Quantification of AdKO versus wt changes in myocardial mRNA expressions after PO represented as average C(t) value fold sham, where sham is set to 1. All values are average of n = 4 to 6 mice per group. * = p < 0.05.</p

List of ECM-related genes analyzed by customized PCR array and their respective Referece Sequence Number.

<p>List of ECM-related genes analyzed by customized PCR array and their respective Referece Sequence Number.</p

Adiponectin is retained in the heart following PO.

<p>(A) Analysis of serum adiponectin by ELISA. Serum was collected at time of euthanization from AdKO or WT mice 2 or 4 weeks following sham or MTAB surgery. Values are represented as mean of 4 to 6 mice per group ± sem. (B) Western blot analysis of cardiac homogenate samples from AdKO or WT mice 2 or 4 weeks following sham or MTAB surgery, quantified in the graph below alongside quantitative real-time PCR analysis of adiponectin mRNA obtained from cardiac homogenates isolated from AdWT mice 2 or 4 weeks following sham or MTAB surgery. Values are represented as average C(t) fold sham, where sham is set to 1. Values are average of n = 3 to 5 mice per group ± SEM.</p

Adiponectin increases collagen synthesis and secretion, and MMP2 activation from cardiac fibroblasts.

<p>Adiponectin stimulation of isolated neonatal cardiac fibroblasts was followed by analysis of: (A) Intracellular pro-collagen synthesis was assessed by ³H-proline incorporation following adiponectin treatment (5 μg/mL) for 6, 24 or 48 h. Data represent mean values ± SEM from n = 3 experiments using 3 wells per group for quantification. Total secreted collagen was measured in fibroblast conditioned media following adiponectin treatment for 6, 24 or 48 h by picrosirius red staining. MMP2 activation was analyzed by gelatin zymography in conditioned media collected from 6, 24 and 48 h adiponectin treated fibroblasts. Data represented as mean arbitrary units ± SEM from n = 7 experiments. (B) Immunofluorescent images of intracellular collagen I (red) and collagen III (green) synthesized in cardiac fibroblasts at 60x magnification. Cells were treated with adiponectin for 6, 24 and 48 h. Representative images from n = 3 experiments are shown. (C) Immunofluorescent images of extracellular collagen I (green) and collagen III (red) secreted from cardiac fibroblasts at 60x magnification. Cells were treated with adiponectin for 3 days. Cell nuclei were also stained with DAPI (blue). Representative images from n = 3 experiments are shown. Below, 3-dimensional stacks of Ad-treated NCFs immunostained for Collagen I and Collagen III were rotated to show relative vertical orientation of nuclei (DAPI) and collagen (green). Arrow head indicates coverslip. (D) Fibroblast proliferation was assessed by ³H-thymidine incorporation following adiponectin treatment for 6, 24, or 48 h. Data represent mean values ± SEM from n = 3 experiments using 3 wells per group for quantification. (E) Fibroblast migration was assessed using the wound-scratch assay following adiponectin treatment for 6 or 24 h. Cell nuclei were stained with DAPI and imaged using confocal microscopy under a 20x objective. Data represent mean values ± SEM from n = 3 experiments using 7–10 images per group for quantification. Representative images are shown in (F). (G) Western blot analysis of cardiac fibroblast cell lysates treated with adiponectin (5 μg/mL) and/or pre-treated with AngII (1 μM). (H) Immunofluorescent staining for αSMA in cardiac fibroblasts treated with adiponectin and/or pre-treated with AngII (1 μM), and imaged using confocal microscopy under a 20x objective.</p

Adiponectin deficiency is associated with increased basal cardiac fibrosis.

<p>Representative scanning electron micrographs of fixed left ventricular samples from Ad KO or wt C57BL/6 mice 2 or 4 weeks following sham or MTAB surgery. Magnifications shown are x1K, 2K or 5K as indicated. Images shown are representative of 5–10 images of n = 4 to 6 mice per group.</p

Quantitative analysis of scanning electron microscopy and histology studies.

<p>Quantitative analysis of collagen fibre diameter (A&B) and vimentin staining (C&D) was assessed as described in methods. (A&B) Five images were taken per mounted tissue and three tissue samples were produced per animal heart (= 15 images/animal heart). (C&D) Five images were taken per stained slide and three slides were produced per animal heart (= 15 images/animal heart). Mean values and standard deviations were calculated form the values obtained for three different animals per experimental condition (n = 3) and values are mean ± SEM.</p

Cellular responses to PO in the myocardium.

<p>WT mice were Sham operated or subject to aortic banding (MTAB) and sacrificed after the indicated time points. Transmural blocks of the left ventricular myocardium were sectioned and immunostained for (A) α-SMA (SMA, blue), desmin (des, red), vimentin (vim, green) and nuclei (DAOI, white) or (B) α-SMA (SMA, green), desmin (des, red), and nuclei (DAOI, blue). Note that exposure time for α-SMA imaging was adjusted in (A) to the high staining intensity in vascular smooth muscle and in (B) to the comparably lower staining intensity in cardiomyocytes overloaded myocardium (~2-times longer exposure). Scale bars: 50 μm.</p