The evolutionarily conserved IGF‐1 signalling pathway is associated with longevity, metabolism, tissue homeostasis, and cancer progression. Its regulation relies on the delicate balance between activating kinases and suppressing phosphatases and is still not very well understood. We report here that IGF‐1 signalling in vitro and in a murine ageing model in vivo is suppressed in response to accumulation of superoxide anions () in mitochondria, either by chemical inhibition of complex I or by genetic silencing of ‐dismutating mitochondrial Sod2. The ‐dependent suppression of IGF‐1 signalling resulted in decreased proliferation of murine dermal fibroblasts, affected translation initiation factors and suppressed the expression of α1(I), α1(III), and α2(I) collagen, the hallmarks of skin ageing. Enhanced led to activation of the phosphatases PTP1B and PTEN, which via dephosphorylation of the IGF‐1 receptor and phosphatidylinositol 3,4,5‐triphosphate dampened IGF‐1 signalling. Genetic and pharmacologic inhibition of PTP1B and PTEN abrogated ‐induced IGF‐1 resistance and rescued the ageing skin phenotype. We thus identify previously unreported signature events with , PTP1B, and PTEN as promising targets for drug development to prevent IGF‐1 resistance‐related pathologies.
New insight into a previously unreported mitochondrial superoxide anion ()‐dependent activation of PTP1B and PTEN with subsequent repression of IGF‐1 signalling, and its implications for skin ageing and other pathologies with aberrant IGF‐1 signalling.
Novel suppressive role of superoxide anions () on IGF‐1 signalling.
Accumulation of mitochondrial activates and induces membrane translocation of ER‐bound PTP1B and cytosolic PTEN.
Inhibition of PTP1B and PTEN rescue IGF‐1 signalling even in the presence of high mitochondrial .
Insulin‐like growth factors (IGFs) play essential roles in the regulation of cell growth, proliferation, stem cell maintenance and synthesis of extracellular matrix proteins (Baker et al, 1993; Le Roith, 1997; Papaconstantinou, 2009; Piecewicz et al, 2012) and—if dysregulated—result in connective tissue and organ atrophy with enhanced ageing or cancer progression (Baker et al, 1993; Pollak et al, 2004; Govoni et al, 2007b; Laviola et al, 2008; Anisimov & Bartke, 2013). During ageing, a gradual decline in circulating insulin‐like growth factor‐1 (IGF‐1) levels beginning in the third and fourth decade of life occurs in humans and in other mammals (Lamberts et al, 1997; Le Roith, 1997; Tatar et al, 2003; Parekh et al, 2010). Reduced IGF‐1 levels and/or impaired IGF‐1 signal transduction may at least in part be responsible for enhanced muscle atrophy (sarcopenia), bone resorption (osteoporosis), and skin atrophy consistently observed in elderly individuals (Gallagher & LeRoith, 2011). Given the significant clinical implications and the current demographic development, advanced knowledge on IGF‐1 signalling and its control at different steps of the signalling cascade is particularly important. Regulation of IGF‐1 signalling occurs rather in integrated signalling networks, which are governed at the level of phosphorylation and dephosphorylation, catalysed by protein kinases and phosphatases, respectively (Pollak et al, 2004; Taguchi & White, 2008). Similar to other growth factors, IGF‐1 signalling is initiated by the autophosphorylation of the IGF‐1 receptor β subunit (IGF‐1Rβ), which is followed by a series of kinase‐dependent phosphorylations of downstream effectors such as phosphoinositide‐3‐kinase (PI3K), AKT (protein kinase B), and p70S6 ribosomal protein kinase (p70S6K)—a prerequisite for synthesis of extracellular matrix—and other proteins. This in conjunction with the IGF‐1‐initiated Ras‐dependent upregulation of cyclin D1 ultimately promotes cell cycle progression and growth essential for overall tissue homeostasis (Pollak et al, 2004; Samani et al, 2007). In addition to the PI3K–AKT axis, IGF‐1 signalling was reported to activate the mitogen‐activated protein kinase (MAPK) pathway, thus exerting its prosurvival effect in many though not all cell lines (Parrizas et al, 1997; Peruzzi et al, 1999; Subramaniam et al, 2005). In fact, IGF‐1 can also inhibit ERK activation in some cell types, including neurons (Subramaniam et al, 2005). Therefore, the effect of IGF‐1 on MAPK and cyclin D1 expression is not uniform for all cell types and most likely depends on the cell type, the nature, magnitude, and duration of the stimulus.
Protein tyrosine phosphatase 1B (PTP1B) was the first identified member of the classical tyrosine‐specific protein phosphatase superfamily (Tonks et al, 1988), which dephosphorylates and thus inactivates the β chain of the IGF‐1R (Buckley et al, 2002). PTP1B is localized at the cytoplasmic site of the endoplasmic reticulum (ER) (Frangioni et al, 1992). After translocation to the plasma membrane, it is endowed with the capacity to dephosphorylate plasma membrane‐associated IGF‐1R (Buckley et al, 2002; Yudushkin et al, 2007) and ligand‐activated IGF‐1R after endocytosis (Eden et al, 2010; Stuible et al, 2010). Similar to the activation of PTP1B, the lipid phosphatase and tensin homologue (PTEN) translocate from the cytosol to the plasma membrane, essential for its activation, and attache to the membrane through its C2 domain (Das et al, 2003; Leslie et al, 2003; Vazquez et al, 2006). Membrane‐bound activated PTEN dephosphorylates membrane‐anchored phosphatidylinositol 3,4,5‐trisphosphate (PIP3), thus opposing PI3K action with subsequent attenuation of IGF‐1 signalling (Maehama & Dixon, 1998).
Several lines of evidence suggest that reactive oxygen species (ROS), in particular H2O2, interfere with insulin/IGF‐1 signalling (Leslie et al, 2003; Houstis et al, 2006; Bashan et al, 2009; Loh et al, 2009; Finkel, 2011; Murphy et al, 2011). Both PTP1B and PTEN are regulated through reversible oxidation and inactivation (Leslie et al, 2003; Salmeen et al, 2003) and thus may be involved in enhanced IGF‐1 signalling. PTP1B and PTEN contain a critical cysteine residue in their active site that exclusively in its reduced state is able to participate in substrate dephosphorylation. In fact, H2O2‐mediated oxidation of cysteine inactivates these phosphatases in vitro (Gough & Cotter, 2011). In addition, it was reported that mice deficient of glutathione peroxidase (Gpx), the key enzyme responsible for H2O2 detoxification, were distinctly protected from high fat‐induced insulin resistance (Loh et al, 2009). This study provides causal evidence for the enhancement of insulin signalling by H2O2 in vivo, suggesting that oxidative inactivation of PTP1B and PTEN stimulates IGF‐1 signalling. In the physiological context, H2O2 is also generated following binding of growth factors to their receptors, thus promoting growth factor signalling by oxidative inactivation of phosphatases (Sundaresan et al, 1995).
By contrast to H2O2, superoxide anion radicals () physiologically occur as a metabolic by‐product during oxidative phosphorylation within mitochondria. Apart from a correlative report (Hoehn et al, 2009), their biology has not been studied in great detail under physiologic and pathologic conditions. An in‐depth knowledge on the role of would, however, be particularly relevant as accumulates in a variety of cells during ageing (Treiber et al, 2011), neurodegeneration (Wu et al, 2010), and conditions such as the metabolic syndrome and diabetes (Kim et al, 2008). In addition, plays a central role in inflammatory conditions (Shishido et al, 1994; Naya et al, 1997; Cheng et al, 1999; MacArthur et al, 2000; Lu et al, 2003) and in chronic non‐healing wounds (Sindrilaru et al, 2011).
Using complementary biochemical and genetic approaches to specifically dissect the effect of and H2O2 on IGF‐1 signalling, we here found that by contrast to the H2O2‐dependent inhibition of the key phosphatases PTP1B and PTEN, accumulation of mitochondrial resulted in marked activation of PTP1B and PTEN, eventually dampening the IGF‐1‐induced signalling cascade at distinct steps of downstream effectors. This leads to subsequent inhibition of murine dermal fibroblast (MDFs) proliferation, changed expression of factors responsible for protein translation initiation, and reduced gene expression of α1 (I), α1 (III), and α2 (I) collagen chains, key features of skin ageing. The ‐dependent IGF‐1 resistance was significantly abrogated by pharmacologic and genetic inhibition of PTP1B and PTEN, suggesting a central role for these phosphatases in the ‐dependent IGF‐1 resistance. This was confirmed in vivo by the rescue of the skin ageing phenotype and IGF‐1 signalling in fibroblast‐specific Sod2‐deficient mice, where PTEN gene was heterozygously deleted in Sod2‐deficient mice. Similarly, pharmacologic PTP1B inhibition in Sod2‐deficient mice showed partial reversal of the impaired IGF‐1 signalling. An advanced understanding of the involvement of distinct ROS species in the regulation of IGF‐1 resistance holds substantial promise for prevention and therapy of age‐related and other pathologies.
Superoxide anions and hydrogen peroxide differentially regulate the IGF‐1/AKT pathway
Murine dermal fibroblasts (MDFs) endogenously produce and release IGF‐1, which via autocrine and paracrine mechanisms stimulate the IGF‐1 signalling cascade (Yakar et al, 1999). To study the effect of different ROS species such as superoxide anion () and hydrogen peroxide (H2O2) on the IGF‐1/AKT axis, MDFs were treated with rotenone, an established inhibitor of complex I of the mitochondrial electron transport chain (Li et al, 2003) or with exogenous H2O2. Inhibition of complex I by rotenone increases electron leakage from this complex to the matrix side, finally leading to partial reduction of oxygen to (Buetler et al, 2004) (Supplementary Fig S1A). As expected, rotenone concentration dependently increased mitochondrial generation in MDFs assessed by MitoSOX, a specific indicator for mitochondrial (Supplementary Fig S1B). We first analysed the phosphorylation of AKT as a critical major downstream signalling component following IGF‐1R activation. Notably, and H2O2 exerted opposite effects on AKT signalling in MDFs, irrespective of the rotenone and H2O2 concentration (Fig 1A) and the time of exposure (Fig 1B). H2O2 at moderate and high concentrations ranging from 10 to 1,000 μM markedly induced phosphorylation of AKT without exerting any effect on total AKT levels (Fig 1A), a finding which is in agreement with published reports (Ushio‐Fukai et al, 1999). Interestingly, rotenone resulted in a concentration‐dependent reduction in AKT activation as shown by reduced phosphorylation of AKT at Ser 473 (pAKT) (Fig 1A). At higher concentrations, both rotenone‐induced and H2O2 led to a marked reduction in the amount of cyclin D1 (Fig 1A and B), which at physiological concentrations is required for cell cycle progression from the G1 phase (Winston et al, 1996). Collagen type I and III synthesis is dependent on IGF‐1/AKT signalling and is mainly controlled at the mRNA level (Gillery et al, 1992). These collagen types produced by MDFs constitute the major extracellular matrix proteins of the skin, which significantly decrease during ageing of murine and human skin (Quan et al, 2010). Interestingly, enhanced generation in MDFs at a rotenone concentration of 500 μM for 3 h inhibited the specific mRNA levels of the α1 and α2 chains of type I collagen and the α1 chain of type III collagen (Fig 1C), while H2O2 at a concentration of 500 μM—parallel to enhancing IGF‐1 signalling—increased α chains of type I and type III collagen‐specific mRNA levels (Fig 1C). Of note, more than 90% of MDFs were viable at rotenone and H2O2 concentrations of 500 μM, indicating that altered collagen synthesis was not related to toxicity (Supplementary Fig S1C). Collectively, these data demonstrate that and H2O2 exert at least partly opposing effects on IGF‐1/AKT signalling in MDFs.
Enhanced superoxide anion concentrations induce partial IGF‐1 resistance
Next, we analysed the IGF‐1R sensitivity to its exogenous ligand in the presence of high concentrations (Fig 2A). Recombinant IGF‐1 (IGF‐1) was used at a concentration of 100 ng/ml to activate the IGF‐1/AKT pathway in MDFs in the presence or absence of rotenone. Addition of IGF‐1 resulted in the activation (phosphorylation) of IGF‐1Rβ, AKT, ribosomal protein S6 (S6), and increased cyclin D1 levels in MDFs (Fig 2B). Notably, rotenone at concentrations of 350 and 500 μM led to a significant inhibition of IGF‐1‐induced activation (phosphorylation) of AKT (pAKT) (Fig 2B), without any alteration of total AKT and IGF‐1Rβ levels. Rotenone treatment markedly inhibited IGF‐1‐mediated IGF‐1Rβ phosphorylation (Fig 2B), suggesting that enhanced concentrations suppressed the IGF‐1/AKT axis at the initial step of IGF‐1‐mediated IGF‐1Rβ activation. IGF‐1 has no effect on activation of ERK in MDFs at least at 60 min post‐stimulation (Fig 2C), although there are reports of ERK activation by IGF‐1 in other cell types (Parrizas et al, 1997; Peruzzi et al, 1999). In fact, IGF‐1 induces the expression of cyclin D1 (Fig 2B), but does not induce the activation of ERK (Fig 2C). The expression of cyclin D1, which is suppressed after IGF‐1 treatment in the presence of rotenone (Fig 2B) very much, suggests that cyclin D1 is not regulated by the IGF‐1R‐induced ERK phosphorylation, but rather by IGF‐1‐induced AKT phosphorylation. The observed rotenone ()‐mediated reduction of cyclin D1 expression is possibly due to the inhibition of other pathways or effectors downstream of IGF‐1R (Winston et al, 1996). Of note, eIF4G, the protein essential for initiation of translation, was decreased, and 4EBP1, the counteracting inhibitor of the initiation process of translation (Kong & Lasko, 2012), was markedly increased in the presence of high concentrations despite IGF‐1 stimulation (Fig 2D). These data imply that steps critically required for translation initiation and protein synthesis might be suppressed by (Fig 2D). In addition, rotenone‐induced was found to suppress IGF‐1‐mediated cell proliferation, as indicated by reduced Ki67 and BrdU labelling (Fig 2E and Supplementary Fig S2). Rotenone alone reduced basal cell proliferation, and additional supplementation with IGF‐1 could only slightly enhance Ki67‐positive cells and BrdU incorporation, indicating that at higher concentrations overwhelmed the cell proliferation stimulatory effect of IGF‐1 (Fig 2E and Supplementary Fig S2). H2O2 at a concentration of 100 μM suppressed cell proliferation (Fig 2E and Supplementary Fig S2), and IGF‐1 only partly attenuated the inhibitory effect of H2O2 on basal cell proliferation (Fig 2E and Supplementary Fig S2). MDFs isolated from Sod2 knockout mice, due to the lack of detoxification, displayed high intracellular concentrations (Treiber et al, 2011). These MDFs revealed a marked reduction in pAKT compared to Sod2 competent murine MDFs (Fig 2F, left panel). Lentivirus vector‐based silencing of Sod2 in MDFs (Sod2 shRNA) also showed higher MitoSox fluorescence indicative of increased concentrations (Supplementary Fig S1D). Following IGF‐1 treatment, Sod2‐silenced MDFs showed partial IGF‐1 resistance with lower pAKT levels when compared to IGF‐1‐treated Sod2 competent control MDFs (co) (Fig 2F, right panel). Rotenone also induced partial IGF‐1 resistance in murine keratinocytes (epidermal skin cells) (Supplementary Fig S3), suggesting that ‐induced IGF‐1 resistance is not restricted to MDFs. These data suggest that enhanced concentrations impair IGF‐1 signalling eventually resulting in IGF‐1 resistance.
In vivo evidence for superoxide anion‐induced partial IGF‐1 resistance
Connective tissue‐specific inducible Sod2‐deficient mice were used to study whether ‐mediated partial IGF‐1 resistance also occurs in vivo. Sod2 floxed mice (Strassburger et al, 2005; Treiber et al, 2011) were crossed with transgenic mice expressing a fusion protein of Cre recombinase and a tamoxifen responsive element under the control of the fibroblast‐specific collagen type I α2 promoter (Col(I)α2‐CreERT mice) (Zheng et al, 2002) to generate Col(I)α2‐CreERT+;Sod2f/f mice (mutant) and Col(I)α2‐CreERT−;Sod2f/f mice (control with competent Sod2 gene). Deletion of Sod2 in the dermal compartment (connective tissue and fibroblast‐rich part) of the skin was performed by administration of 4‐OH tamoxifen (Supplementary Fig S4A and B). Both control and mutant mice were intraperitoneally (i.p.) injected either with 1 mg/kg recombinant IGF‐1 or with normal saline. Indeed, IGF‐1 treatment significantly induced the AKT phosphorylation (Ser 473) in the skin of control mice compared to saline‐treated control mice as shown by immunofluorescence staining of skin sections and Western blot analyses of skin lysates (Fig 3A and B). Strikingly, injection of IGF‐1 only slightly induced AKT phosphorylation in the skin of mutant mice (with at higher concentrations) compared to saline‐treated mutant mice. Of note, the extent of AKT phosphorylation was markedly decreased in the fibroblast‐rich dermis but not in the adipocyte‐containing subcutaneous layer of IGF‐1‐injected mutant mice compared to IGF‐1‐treated control mice (Fig 3A). The maximum stimulatory effect of IGF‐1 was found 15 min after injection, which was no longer detectable at 60 min after IGF‐1 injection (Fig 3B and Supplementary Fig S4C). As higher concentrations were selectively attained by fibroblast‐specific deletion of Sod2 (Treiber et al, 2011), these data underscore the specificity of our model and a previously unreported critical role of increased concentrations in suppressing the IGF‐1/AKT axis in vivo.
IGF‐1 resistance is due to enhanced PTP1B and PTEN phosphatase activity
As we have observed that rotenone inhibited IGF‐1‐induced autophosphorylation of tyrosine residues (Tyr 1135 and 1136) of IGF‐1Rβ (Fig 2B), we further explored the possibility whether the activation of specific tyrosine phosphatases is responsible for the observed IGF‐1Rβ dephosphorylation. To achieve this, we investigated the role of key phosphatases including PTP1B, PTEN, and PP2A involved in the regulation of IGF‐1 signalling (Maehama & Dixon, 1998; Millward et al, 1999; Buckley et al, 2002). No significant change was observed in basal expression levels of PTP1B, PTEN, and PP2A phosphatases of MDFs subjected to different rotenone concentrations compared with non‐treated MDFs (Fig 4A). Also PTP1B activity was not enhanced in whole‐cell lysates prepared from rotenone‐treated MDFs (Fig 4B). Interestingly, increased activities of PTP1B bound to its substrate (IGF‐1Rβ) and PTEN occurred in rotenone‐treated MDFs compared with non‐treated MDFs (Fig 4C and D), while no change was observed in PP2A activity (Fig 4E). This suggests an important role for PTP1B and PTEN in the ‐mediated suppression of the IGF‐1/AKT axis. As membrane localization of these phosphatases is an essential prerequisite for the inactivation of IGF‐1Rβ and downstream effectors of the IGF‐1 signalling pathway (Buckley et al, 2002; Yudushkin et al, 2007), we analysed whether upon rotenone or H2O2 treatment, PTP1B and PTEN translocate to the plasma membrane from the endoplasmic reticulum and cytoplasm, respectively. Notably, PTP1B and PTEN were recruited to the plasma membrane when MDFs were subjected to rotenone (Fig 5A and B and corresponding inserts), whereas no such translocation to the plasma membrane was observed in non‐treated control and H2O2‐treated cells (Fig 5A and B) Up to fourfold more PTP1B and PTEN were translocated to the membrane of rotenone‐treated cells (Fig 5A and B, right panels). Western immunoblot analyses further confirmed the enrichment of PTEN and PTP1B in the membrane fraction of rotenone‐treated MDFs compared to non‐treated controls and H2O2‐treated MDFs (Fig 5C and Supplementary Fig S5A and B). Using co‐immunoprecipitation (Co‐IP) experiments with IGF‐1Rβ antibodies, a physical interaction of PTP1B with IGF‐1Rβ was found after rotenone‐induced stress (Fig 5D), and, as earlier shown, IGF‐1Rβ‐bound PTP1B displayed higher phosphatase activity in rotenone‐treated MDFs (Fig 4D). Furthermore, transient overexpression of Sod2 with increased scavenging capacity (Supplementary Fig S1E) significantly inhibited rotenone‐induced membrane translocation of PTP1B and PTEN in MDFs (Fig 6A and B), suggesting a causal contribution of enhanced concentrations in the translocation of these two phosphatases to the plasma membrane. The fact that overexpression of Sod2 also improved IGF‐1 sensitivity in rotenone‐treated MDFs further supports a critical role of enhanced concentrations in the activation of these phosphatases with subsequent IGF‐1 suppression (Fig 6C). Stimulated by earlier reports that the phosphorylation of serine and threonine residues in the C‐terminal domain of PTEN restricts it to the cytoplasm, while dephosphorylation favours its membrane localization and activation (Vazquez et al, 2006; Rahdar et al, 2009), we studied whether high concentrations had any impact on the phosphorylation status of PTEN. For this purpose, PTEN was immunoprecipitated from the cytoplasmic and membrane fractions of vehicle or rotenone‐treated MDFs followed by immunoblotting with antibodies against phospho‐serine, phospho‐threonine, and phospho‐tyrosine. Interestingly, a membrane translocation phosphorylation pattern was found with reduced serine phosphorylation in cytoplasmic and membrane PTEN in rotenone‐treated MDFs compared to vehicle‐treated MDFs, while no threonine phosphorylation was detected in the membrane fraction of PTEN in both rotenone‐treated and control MDFs (Supplementary Fig S6). In the cytoplasmic PTEN fraction, rotenone‐treated MDFs showed lower threonine phosphorylation (Supplementary Fig S6). Of note, enhanced tyrosine phosphorylation in the membrane PTEN pool and reduced tyrosine phosphorylation in the cytosolic PTEN were found in rotenone‐treated compared to vehicle‐treated MDFs (Supplementary Fig S6). These data indicate that the ‐dependent PTEN translocation from the cytoplasm to the membrane correlates with specific post‐translational modifications, the precise nature of which awaits elucidation. Taken together, the ‐dependent PTP1B activation mediates IGF‐1/AKT suppression at the step of IGF‐1Rβ dephosphorylation, while PTEN recruitment and activation at the plasma membrane enhance the dephosphorylation of its phospholipid substrate PIP3 to PIP2, thus suppressing the phosphorylation (activation) of AKT.
Inhibition of PTP1B and PTEN rescues the superoxide anion‐induced IGF‐1 resistance
To further strengthen the causal role of PTP1B and PTEN in the ‐dependent IGF‐1 resistance, we used pharmacological inhibitors and specific gene silencing and studied the phosphorylation state of AKT, a key step in IGF‐1 downstream signalling. Notably, the PTP1B inhibitor (3‐(3,5‐Dibromo‐4‐hydroxy‐benzoyl)‐2‐ethyl‐benzofuran‐6‐sulfonicacid‐(4‐(thiazol‐2‐ylsulfamyl)‐phenyl)‐amide) at a concentration of 50 μM in part rescued the suppression of AKT phosphorylation and reduced eIF4G levels in MDFs exposed to recombinant IGF‐1 in the presence of rotenone (Fig 7A). In addition, the PTP1B inhibitor opposed rotenone‐induced suppression of IGF‐1Rβ phosphorylation in MDFs stimulated with IGF‐1 (Supplementary Fig S7). Similarly, the suppressive action of high concentrations on IGF‐1‐induced AKT phosphorylation was rescued by 150 nM VO‐OHpic, a vanadium‐based specific PTEN inhibitor (Rosivatz et al, 2006) (Fig 7B). In addition, silencing of PTP1B or PTEN using lentivirus‐mediated shRNA expression significantly alleviated the suppressed activation state (phosphorylation) of AKT in MDFs treated with IGF‐1 in the presence of rotenone (Fig 7C). These data strongly suggest that enhanced activation of PTP1B and PTEN is causally involved in the ‐dependent IGF‐1 resistance. To further explore whether our in vitro observations may also have relevance in vivo for tissue homeostasis, we used a genetic approach. Deletion of Sod2 in fibroblasts of the connective tissue‐rich skin resulted in skin atrophy, a key feature of ageing, with reduced thickness of all the skin layers (Fig 8A and B). Heterozygous deficiency of PTEN in the Sod2‐deficient fibroblasts as achieved by crossing PTEN floxed mice with Col(I)α2‐CreERT transgenic and Sod2 floxed mice (Col(I)α2CreERT+;Sod2f/f) followed by activation of CreERT with tamoxifen treatment (Supplementary Fig S8A and B) rescued skin atrophy/ageing in the double mutant mice (Col(I)α2‐CreERT+;Sod2f/f;PTENf/+) compared with Sod2 mutant mice (Col(I)α2CreERT+;Sod2f/f;PTEN+/+) (Fig 8A and B). Of note, genetic heterozygous deficiency of PTEN with reduced PTEN expression or inhibition of PTP1B by specific inhibitor also attenuated the IGF‐1 resistance in vivo in a Sod2‐deficient murine ageing model (Fig 8C and D). Accordingly, heterozygous PTEN deletion rescued IGF‐1‐stimulated phosphorylation of AKT in the skin of fibroblast‐specific Sod2‐deficient mice (Fig 8C and D). Similarly, continuous inhibition of PTP1B with its specific inhibitor at a dose of 15 mg/kg for 5 days also rescued IGF‐1‐induced phosphorylation of AKT in the skin of fibroblast‐specific Sod2‐deficient mice (Fig 8C and D). These data show that enhanced ‐ concentrations in dermal fibroblasts in the skin in situ—via PTEN and PTP1B activation—dampen the IGF‐1 signalling also in vivo and most likely contribute to skin ageing/atrophy.
The major finding of this report is that accumulation of mitochondrial led to IGF‐1 resistance by tilting the delicate balance of regulatory kinases and phosphatases towards enhanced activities of PTP1B and PTEN, key phosphatases of the IGF‐1 signalling pathway. Enhanced activities of PTEN and PTP1B were associated with their membrane translocation. To the best of our knowledge, this finding identifies a previously unreported role of PTP1B and PTEN as molecular links mediating repression of IGF‐1 signalling at high superoxide anion () concentrations in the mammalian system (Fig 9). The resulting IGF‐1 resistance led to reduced fibroblast proliferation, marked suppression of specific α1 (I), α2 (I), and α1 (III) collagen chain mRNA levels, and changed expression of key factors responsible for translation initiation, collectively representing signature events of ageing and organ atrophy. Notably, genetic and pharmacological inhibition of PTP1B and PTEN alleviated IGF‐1 resistance under conditions of enhanced concentrations underlining their causal role and making PTP1B and PTEN potential targets for drug development. The finding that ImpL2, the Drosophila ortholog of insulin‐like growth factor‐binding protein 7 (IGFBP7), antagonizes insulin signalling in response to mitochondrial complex I perturbation (Owusu‐Ansah et al, 2013) further underscores an evolutionarily conserved linkage between mitochondrial dysfunction and aberrant insulin signalling.
Enhanced concentrations have been reported in several physiological and pathological inflammatory conditions (Shishido et al, 1994; Naya et al, 1997; Cheng et al, 1999; MacArthur et al, 2000; Lu et al, 2003; Liu et al, 2004; Sindrilaru et al, 2011). In addition, enhanced concentrations were also detected in senescent dermal fibroblasts in vitro and in the fibroblast from connective tissue‐specific Sod2‐deficient mice (Treiber et al, 2011) and—via dampening IGF‐1 signalling—may contribute to ageing phenotypes of connective tissue‐rich organs such as skin, bone, and muscle. This notion is consistent with the finding that these organs are particularly dependent on balanced IGF‐1 signalling to maintain tissue homeostasis (Govoni et al, 2007a,b). Enhanced concentrations, as shown herein, suppressed fibroblast proliferation and the synthesis of interstitial collagen chains, the major extracellular matrix proteins of the skin. In fact, suppression of collagen synthesis and deposition as well as reduced proliferation constitute key molecular events in skin ageing (Fisher et al, 2008; Quan et al, 2010). Of note, IGF‐1 was earlier reported to stimulate collagen synthesis and fibroblast growth (Gillery et al, 1992), and—if IGF‐1 is suppressed by ‐induced oxidative stress—results in skin ageing and atrophy. In addition, chronic venous leg ulcers and diabetic ulcers reveal a persistent inflammation with enhanced activation of pro‐inflammatory macrophages, and an unrestrained release of , which at persistent high concentrations, overruns the ‐dismutating capacity of non‐enzymatic and enzymatic antioxidants (Sindrilaru et al, 2011). Hence, is the prevailing reactive oxygen species in chronic wounds and according to the herein reported data may dampen the signalling of IGF‐1 and other growth factors and impair tissue repair processes. This notion is consistent with previous reports that many clinical trials with recombinant growth factors for the treatment of chronic wounds either failed or required extremely high concentrations of recombinant growth factors for substitution, indicating growth factor resistance of chronic wounds (Bitar & Al‐Mulla, 2012). This poor outcome may at least in part be due to an ‐dependent enhanced phosphatase activation with subsequent attenuation of growth factor signalling.
In this study, we were interested in the role of distinct ROS such as mitochondria‐derived and H2O2 on IGF‐1 sensitivity and downstream signalling. Strikingly, as opposed to the established H2O2‐mediated enhanced insulin/IGF‐1 sensitivity (Loh et al, 2009), we found that high concentrations following rotenone‐mediated inhibition of mitochondrial complex I or by silencing of ‐dismutating mitochondrial Sod2 resulted in IGF‐1 resistance. Indeed, we identified enhanced activities and membrane localization of PTP1B and PTEN following high load, and interestingly, this effect was abolished by overexpression of Sod2. IGF‐1 resistance was also observed in Sod2‐deficient fibroblasts with significant accumulation of mitochondrial , but not of H2O2 as proven by ROS‐specific quantitative techniques (Treiber et al, 2011). Moreover, scavenging of mitochondrial by the overexpression of Sod2 in the mitochondria of rotenone‐treated murine dermal fibroblasts attenuated the observed IGF‐1 resistance. Rotenone may not only suppress complex I of the electron transfer chain to enhance intramitochondrial concentrations. From a previous publication, rotenone has been implicated in enhancing the NADPH oxidase in microglial cells of the brain with enhanced release into the extracellular space with subsequent neuron degeneration (Gao et al, 2003). Even though we cannot exclude this or other rotenone effects in our study, we provide consistent evidence that the rotenone‐mediated inhibition of complex I with accumulation of high concentrations or accumulation by silencing of ‐dismutating mitochondrial Sod2 resulted in IGF‐1 resistance.
Our conclusion to have identified a novel route of signalling with an ‐dependent increase of PTP1B and PTEN activity is based on several lines of evidence. First, in the case of PTP1B, we found a translocation of PTP1B from the ER to plasma membranes and co‐localization with IGF‐1Rβ. This translocation is indicative of PTP1B activation (Buckley et al, 2002; Yudushkin et al, 2007). Secondly, using co‐immunoprecipitation, a physical interaction of PTP1B with the membrane‐associated IGF‐1Rβ and higher phosphatase activity of the IGF‐1Rβ‐bound PTP1B fraction was observed further supporting our hypothesis and suggesting a key role of PTP1B in ‐driven repression of IGF‐1 signalling. Finally, inhibition of PTP1B either by a pharmacological inhibitor or through shRNA‐mediated knockdown reversed the ‐induced IGF‐1 resistance. The conclusion that PTEN activity was also increased at high concentrations is supported by the following results. First, translocation of PTEN from the cytosol to the plasma membrane indicating PTEN activation (Das et al, 2003; Vazquez et al, 2006; Rahdar et al, 2009) was consistently observed in fibroblasts exposed to enhanced but not to enhanced H2O2 concentrations. Second, higher activity of PTEN was found in rotenone‐treated MDFs. Third, the PTEN‐specific inhibitor, VO‐OHpic, significantly attenuated the ‐dependent suppression of IGF‐1 signalling. VO‐OHpic is a highly selective inhibitor for PTEN and only inhibits other PTPs at concentrations within the high micromolar range (Rosivatz et al, 2006). Fourth, similar results were also observed upon shRNA‐mediated PTEN silencing. Importantly, partial deletion of PTEN and inhibition of PTP1B in Sod2‐deficient mice also rescued the IGF‐1 resistance and the atrophy/ageing skin phenotype. Thus, repression of IGF‐1 signalling by a high load is predominantly mediated by the active participation of PTP1B and PTEN. Taken together, our results unify and explain earlier reports on increased IGF‐1 resistance upon ROS induction (Tiganis, 2011; Johnson & Olefsky, 2013) by specifying that enhanced concentrations are largely responsible for repression of IGF‐1 signalling and that the ‐mediated IGF‐1 resistance is mainly due to aberrant PTP1B and PTEN translocation and activation. The precise (Vazquez et al, 2006; Rahdar et al, 2009) mechanism though of PTP1B and PTEN translocation in response elevated concentrations from the cytoplasm to the plasma membrane needs to be further elucidated.
As to the question how increased concentrations in the mitochondria transfer the signal to the cytoplasm, it is possible that enhanced mitochondrial concentrations lead to modifications of kinases, which shuttle between the mitochondria and the plasma membrane exerting phosphorylation and activation of PTP1B and PTEN—the nature of these anticipated kinases remains, however, elusive. Alternatively, longer‐lived lipid peroxidation intermediates may transmit the ‐induced signals from the mitochondria to cytosolic and/or membrane‐bound PTP1B/PTEN. Notably, is able to initiate lipid peroxidation by itself, or after reacting with nitric oxide (NO) to form peroxynitrite (ONOO−) (Buetler et al, 2004; Bashan et al, 2009). A recent report suggests that can escape mitochondria and enter the cytosol through mitochondrial voltage‐dependent anion channel (Lustgarten et al, 2012).
Regarding the question why evolution has promoted the development of an ‐dependent partial IGF‐1 resistance, it is important to consider that increased concentrations are extremely noxious to cells. ‐mediated repression of IGF‐1 signalling through PTP1B and PTEN activation might have salutary effects on cells with perturbed mitochondria as is the case in type 2 diabetes mellitus, ageing, impaired wound healing, and neurodegenerative disease. In this context, it can act as an oxidant defence mechanism that protects cells by attenuating further ROS generation from increased metabolic or anabolic activity. Also, it can help to promote clearance of damaged cells by apoptosis or by other mechanisms. In fact, lower levels of phosphorylated AKT enhance apoptosis (Franke et al, 1997). Thus, ‐dependent PTP1B and PTEN activation with the induction of IGF‐1 resistance may serve as a reliable guardian to protect cells and tissues from ‐driven oxidative stress and its deleterious consequences.
In summary, we uncovered a mechanism by which enhanced released from dysfunctional mitochondria causes IGF‐1 resistance, thereby repressing IGF‐1 signalling in mammalian cells. Our studies provide insight into a previously undescribed ‐dependent activation of PTP1B and PTEN with subsequent repression of IGF‐1 signalling. Targeting these key players may hold promise for the development of novel therapies for age‐related pathologies with aberrant IGF‐1 signalling such as type 2 diabetes, skin atrophy, osteoporosis, impaired wound healing, and neurodegenerative disease. Unquestionably, future studies should address detailed molecular interactions and identify additional intermediate signalling molecules engaged in ‐dependent repression of IGF‐1 signalling. In addition, it will be exciting to further explore whether—in addition to the IGF‐1 dampening—enhanced concentrations may affect other receptor tyrosine kinases involved in tissue homeostasis.
Materials and Methods
An inducible connective tissue‐specific Sod2‐deficient mouse model was used for in vivo experiments. This mouse line (Col(I)α2‐CreERT+;Sod2f/f) was generated by crossing Col(I)α2‐CreERT transgenic mice (Zheng et al, 2002) with Sod2 floxed mice (Strassburger et al, 2005; Treiber et al, 2011). The generation of Sod2 and PTEN double‐mutant mice (Col(I)α2‐CreERT+;Sod2f/f;PTENf/+) was performed by crossing Sod2‐deficient mice (Col(I)α2‐CreERT+;Sod2f/f) with PTEN flox mice (PTENf/f) (Groszer et al, 2001). All these mice were backcrossed to C57BL/6J for at least 10 generations and were maintained in the Animal facility of University of Ulm with 12 h light–dark cycle and SPF condition. All the animal experiments were approved by the animal ethical committee (Regierungspräsidium Tübingen, Germany). The genotyping of the mice was performed using standard PCR techniques. The sequences of the primers used in PCR‐based genotyping are summarized in Supplementary Table S1. In brief, DNA from the tail tip of an individual mouse was purified using a commercial kit (Easy DNA kit, Invitrogen). The purified DNA was later dissolved in TE and used for PCR amplification. The PCR products were run in QIAxcel Advance system (Qiagen) using the program AM320 and then documented digitally. To initiate the activation of CreERT and deletion of Sod2 and PTEN, newborn mice (10 days old) were orally administered with 50 μl of 1 mg/ml of 4‐OH tamoxifen (Sigma) suspended in 0.5% methylcellulose and 0.1% Tween‐80 (Sigma) for five alternate days. When the mice (both male and female) were 21–25 days old, the mice were subcutaneously implanted with single 60‐day release 4‐OH tamoxifen pellets (Innovative Research of America). At the age of 65–70 days, mice of both sexes were intraperitoneally (i.p.) injected either with 100 μl of 1 mg/ml recombinant IGF‐1 in normal saline or with equal volume of normal saline. Mice were sacrificed 15 or 60 min post‐injection, and skin tissues were collected and processed for paraffin embedding and protein lysate preparation.
Isolation and culture of murine dermal fibroblasts
Murine dermal fibroblasts (MDFs) were isolated from ear skin of young mice and cultured as previously described (Treiber et al, 2011).
Silencing by shRNA
Silencing of Sod2, PTEN, and PTP1B was performed using lentivirus vector‐based shRNA clones from the RNAi consortium (TRC, Broad Institute) (details in Supplementary Methods).
Serum starvation and IGF‐1 stimulation
Murine dermal fibroblasts were washed with PBS and serum‐starved for 14 h before stimulation. Stimulation with recombinant mouse IGF‐1 (ProSpec) was performed at 100 ng/ml concentration for indicated time points.
Membrane, cytosolic, and nuclear fractionation
Murine dermal fibroblasts were harvested, and then the membrane and cytosolic extracts were prepared using the Mem‐PER extraction reagents according to the manufacturer's instructions (Thermo Scientific). Thereafter, 30 μg of cytosolic lysate or membrane extracts was subjected to Western blot analyses.
Activity assay of phosphatases
Phosphatases activity assays were performed in immunoprecipitated protein samples (details in Supplementary Methods).
Immunofluorescence staining and analyses
Immunofluorescence staining was performed as previously described (Treiber et al, 2011).
Cloning and overexpression
Open reading frame (ORF) of mouse Sod2 was PCR‐amplified from its cDNA IMAGE clones (BC010548), and cloned in pcDNA3.1 (Invitrogen) (details in Supplementary Methods).
The viability of murine dermal fibroblasts was measured 3, 6, 12, and 24 h after incubation with rotenone, H2O2, PTP1B inhibitor, and VO‐OHpic using 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) (Sigma) (details in Supplementary Methods).
All data were calculated using Graphpad Prism (GraphPad Software) or Sigmaplot (Systat Software Inc) and presented as mean ± standard error of mean (SEM). Statistical significance (P‐value) was calculated using either one‐way ANOVA, followed by Bonferroni correction for comparing the difference between more than two samples or by unpaired two‐tailed Student's t‐test for comparing the difference between two samples. For two‐tailed t‐test, the exact P‐values and for ANOVA, in multiple comparison test (Bonferroni), either the exact P‐value (when P > 0.001, but < 0.05) or the highest P‐value (P < 0.001) were presented. The exact test which was used and the exact P‐value of comparison are presented in the respective figure legends and figures, respectively.
The paper explained
The evolutionarily conserved IGF‐1 signalling pathway is associated with longevity, metabolism, tissue homeostasis, and cancer progression. Its action is tightly regulated in multiple steps of activation and inhibition and, when dysregulated, results in organ atrophy with enhanced ageing or cancer progression. During ageing, reduced growth and regeneration occur, which lead to organ and skin atrophy characterized by wrinkle formation, reduced tensile strength of the skin, and impaired wound healing. A reduced function of IGF‐1 has long been postulated to inhibit cell proliferation and synthesis of extracellular matrix proteins like collagens in ageing organs; however, the exact mechanisms of IGF‐1 suppression are still poorly understood. Of note, the gradual increase in oxidative stress is now established during the ageing process and several pathologies associated with ageing and oxidative stress in elderly individuals. Here, we addressed the impact of oxidative stress on IGF‐1 function, its signalling, and on cellular key features of skin ageing. The detailed understanding of the IGF‐1 action during oxidative stress in skin ageing and other oxidative stress‐related pathologies is of high clinical importance as it holds promise for the development of preventive and/or therapeutic strategies aiming at the delay and reversal of declining skin function.
Of note, distinct entities of reactive oxygen species modulate IGF‐1 function. Hydrogen peroxide (H2O2) activates IGF‐1 signalling by inhibiting distinct phosphatases involved in IGF‐1 suppression. By contrast, accumulation of mitochondrial as occurs in ageing skin inactivates IGF‐1 signalling. Increasing mitochondrial either by chemical means (rotenone treatment) or by genetic silencing of Sod2, the enzyme which normally detoxifies ‐ in murine dermal fibroblasts, leads to the activation and membrane translocation of the two phosphatases PTP1B and PTEN. PTP1B activation and membrane translocation, in fact, lead to its close association with the membrane‐anchored IGF‐1 receptor; this in turn promotes the dephosphorylation (inactivation) of the IGF‐1 receptor (IGF‐1R β subunit) with an almost complete abrogation of IGF‐1 signalling and subsequent suppressed growth of skin fibroblasts. In addition, PTEN dephosphorylates PIP3 to PIP2 at a later step of IGF‐1 signalling, leading to inhibition of AKT phosphorylation (activation), a key step essential for cell growth. Attenuation of ‐mediated inhibition of IGF‐1 signalling and IGF‐1 resistance by overexpression of Sod2 further supports the causal contribution of increased concentrations to IGF‐1 resistance. Inhibition of PTP1B and PTEN by specific chemical inhibitors as well as silencing by shRNA in vitro significantly rescues IGF‐1 resistance even in the presence of higher mitochondrial . Moreover, deletion of PTEN and inhibition of PTP1B in Sod2‐deficient mice also attenuate IGF‐1 resistance in vivo. Finally, deletion of PTEN rescues the skin phenotype of Sod2‐deficient mice.
Our novel finding is that different entities of reactive oxygen species, such as H2O2 and , exert opposite effects on the IGF‐1 signalling pathway. Our studies provide insight into a previously undescribed ‐dependent activation of PTP1B and PTEN with subsequent repression of IGF‐1 signalling. Targeting these key players may hold substantial promise for the development of novel therapies for age‐related pathologies with aberrant IGF‐1 signalling such as type 2 diabetes, skin atrophy, osteoporosis, impaired wound healing, neurodegeneration, and cancer.
KS and PMa designed and carried out the experiments and wrote the initial version of the manuscript. LK, PMe, AB and NT technically supported this work. TL was involved in lentiviral works. SK, MW, and HG were involved in designing and technically supporting the experiments. KS‐K designed the experiments and wrote the manuscript.
Conflict of interest
The authors declare that they have no conflict of interest.
KS‐K is supported by the German Research Foundation (DFG, SCHA411/15‐2) within the Clinical Research Group KFO142 “Cellular and Molecular Mechanisms of Ageing – From Mechanisms to Clinical Perspectives”, by the Graduate Training Centre GRK 1789 “Cellular and Molecular Mechanisms in Ageing (CEMMA)”, and the Förderlinie Perspektivförderung “Zelluläre Entscheidungs‐ und Signalwege bei der Alterung” of the Ministerium für Wissenschaft, Forschung und Kunst Baden‐Württemberg, Germany.
FundingGerman Research Foundation (DFG) SCHA411/15‐2
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- © 2014 The Authors. Published under the terms of the CC BY 4.0 license