Christine Wai

Hyperglycemia Enhances IGF-I–Stimulated Src Activation via Increasing Nox4-Derived Reactive Oxygen Species in a PKCζ-Dependent Manner in Vascular Smooth Muscle Cells

IGF-I–stimulated sarcoma viral oncogene (Src) activation during hyperglycemia is required for propagating downstream signaling. The aim of the current study was to determine the mechanism by which hyperglycemia enhances IGF-I–stimulated Src activation and the role of NADPH oxidase 4 (Nox4) and protein kinase C ζ (PKCζ) in mediating this response in vascular smooth muscle cells (VSMCs). Nox4 expression was analyzed in VSMCs exposed to hyperglycemia. The role of Nox4-derived reactive oxygen species (ROS) in IGF-I–stimulated Src activation was investigated via knockdown of Nox4. Different isoforms of PKC were screened to investigate their role in hyperglycemia-induced Nox4. The oxidation of Src was shown to be a prerequisite for its activation in response to IGF-I during hyperglycemia. Hyperglycemia induced Nox4, but not Nox1, and p22 phagocyte oxidase (p22phox) expression and IGF-I stimulated Nox4/p22phox complex formation, leading to increased ROS generation. Knockdown of Nox4 prevented ROS generation and impaired the oxidation and activation of Src in response to IGF-I, whereas knockdown of Nox1 had no effect. PKCζ was shown to mediate the hyperglycemia-induced increase in Nox4 expression. The key observations in cultured VSMCs were confirmed in the diabetic mice. Nox4-derived ROS is responsible for the enhancing effect of hyperglycemia on IGF-I–stimulated Src activation, which in turn amplifies IGF-I–linked downstream signaling and biological actions.

Insulin-Like Growth Factor (IGF) Binding Protein 2 Functions Coordinately with Receptor Protein Tyrosine Phosphatase β and the IGF-I Receptor To Regulate IGF-I-Stimulated Signaling

ABSTRACT Insulin-like growth factor I (IGF-I) is a mitogen for vascular smooth muscle cells (VSMC) and has been implicated in the development and progression of atherosclerosis. IGF binding proteins (IGFBPs) modify IGF-I actions independently of IGF binding, but a receptor-based mechanism by which they function has not been elucidated. We investigated the role of IGFBP-2 and receptor protein tyrosine phosphatase β (RPTPβ) in regulating IGF-I signaling and cellular proliferation. IGFBP-2 bound RPTPβ, which led to its dimerization and inactivation. This enhanced PTEN tyrosine phosphorylation and inhibited PTEN activity. Utilization of substrate trapping and phosphatase-dead mutants showed that RPTPβ bound specifically to PTEN and dephosphorylated it. IGFBP-2 knockdown led to decreased PTEN tyrosine phosphorylation and decreased AKT Ser473 activation. IGFBP-2 enhanced IGF-I-stimulated VSMC migration and proliferation. Analysis of aortas obtained from IGFBP-2−/− mice showed that RPTPβ was activated, and this was associated with inhibition of IGF-I stimulated AKT Ser473 phosphorylation and VSMC proliferation. These changes were rescued following administration of IGFBP-2. These findings present a novel mechanism for coordinate regulation of IGFBP-2 and IGF-I signaling functions that lead to stimulation of VSMC proliferation. The results have important implications for understanding how IGFBPs modulate the cellular response to IGF-I.

Recruitment of Nox4 to a plasma membrane scaffold is required for localized reactive oxygen species generation and sustained Src activation in response to insulin-like growth factor-I.

Background: Nox4-derived ROS is required for Src oxidation and activation. Results: Grb2 recruits Nox4 to the scaffold protein SHPS-1, and Nox4 colocalization with Src on SHPS-1 allows Nox4 to activate Src. Conclusion: Nox4 recruitment to SHPS-1 is essential for sustained Src activation and cell proliferation. Significance: This is the first report that localized ROS generation mediates growth factor signaling in vitro and in vivo. Nox4-derived ROS is increased in response to hyperglycemia and is required for IGF-I-stimulated Src activation. This study was undertaken to determine the mechanism by which Nox4 mediates sustained Src activation. IGF-I stimulated sustained Src activation, which occurred primarily on the SHPS-1 scaffold protein. In vitro oxidation experiments indicated that Nox4-derived ROS was able to oxidize Src when they are in close proximity, and Src oxidation leads to its activation. Therefore we hypothesized that Nox4 recruitment to the plasma membrane scaffold SHPS-1 allowed localized ROS generation to mediate sustained Src oxidation and activation. To determine the mechanism of Nox4 recruitment, we analyzed the role of Grb2, a component of the SHPS-1 signaling complex. We determined that Nox4 Tyr-491 was phosphorylated after IGF-I stimulation and was responsible for Nox4 binding to the SH2 domain of Grb2. Overexpression of a Nox4 mutant, Y491F, prevented Nox4/Grb2 association. Importantly, it also prevented Nox4 recruitment to SHPS-1. The role of Grb2 was confirmed using a Pyk2 Y881F mutant, which blocked Grb2 recruitment to SHPS-1. Cells expressing this mutant had impaired Nox4 recruitment to SHPS-1. IGF-I-stimulated downstream signaling and biological actions were also significantly impaired in Nox4 Y491F-overexpressing cells. Disruption of Nox4 recruitment to SHPS-1 in aorta from diabetic mice inhibited IGF-I-stimulated Src oxidation and activation as well as cell proliferation. These findings provide insight into the mechanism by which localized Nox4-derived ROS regulates the sustained activity of a tyrosine kinase that is critical for mediating signal transduction and biological actions.

Insulin-like growth factor-binding protein-2 is required for osteoclast differentiation.

Global deletion of the Igfbp2 gene results in the suppression of bone turnover. To investigate the role of insulin‐like growth factor‐binding protein‐2 (IGFBP‐2) in regulating osteoclast differentiation, we cultured Igfbp2−/− bone marrow cells and found a reduction in the number of osteoclasts and impaired resorption. Addition of full‐length IGFBP‐2 restored osteoclast differentiation, fusion, and resorption. To determine the molecular domains of IGFBP‐2 that were required for this effect to be manifest, Igfbp2−/− bone marrow cells were transfected with constructs in which the heparin‐binding (HBD) or the IGF‐binding domains of IGFBP‐2 were mutated. We found that both domains were necessary for osteoclastogenesis because expression of the mutated forms of either domain failed to support the formation of functionally mature osteoclasts. To discern the mechanism by which IGFBP‐2 regulates osteoclast formation, PTEN abundance and phosphorylation status as well as AKT responsiveness to IGF‐I were analyzed. Igfbp2−/− cells had elevated levels of PTEN and phospho‐PTEN compared with controls. Expression of wild‐type IGFBP‐2 reduced the level of PTEN to that of wild‐type cells. Cells expressing the IGF‐binding mutant showed suppression of PTEN and phospho‐PTEN equivalent to the wild‐type protein, whereas those expressing the IGFBP‐2 HBD mutant showed no PTEN suppression. When the ability of IGF‐I to stimulate AKT activation, measured by Thr308 and Ser473 phosphorylation, was analyzed, stimulation of Ser473 in response to IGF‐I in preosteoclasts required the presence of intact IGFBP‐2. This effect was duplicated by the addition of a CK2 inhibitor that prevents the phosphorylation of PTEN. In contrast, in fully differentiated osteoclasts, stimulation of Thr308 phosphorylation required the presence of intact IGFBP‐2. We conclude that IGFBP‐2 is an important regulator of osteoclastogenesis and that both the heparin‐ and the IGF‐binding domains of IGFBP‐2 are essential for the formation of fully differentiated and functional osteoclasts.

IGFBP‐2 Directly Stimulates Osteoblast Differentiation

Insulin‐like growth factor binding protein 2 (IGFBP‐2) is important for acquisition of normal bone mass in mice; however, the mechanism by which IGFBP‐2 functions is not defined. These studies investigated the role of IGFBP‐2 in stimulating osteoblast differentiation. MC‐3T3 preosteoblasts expressed IGFBP‐2, and IGFBP‐2 knockdown resulted in a substantial delay in osteoblast differentiation, reduced osteocalcin expression and Alizarin red staining. These findings were replicated in primary calvarial osteoblasts obtained from IGFBP‐2−/− mice, and addition of IGFBP‐2 rescued the differentiation program. In contrast, overexpression of IGFBP‐2 accelerated the time course of differentiation as well as increasing the total number of differentiating cells. By day 6, IGFBP‐2–overexpressing cells expressed twice as much osteocalcin as control cultures and this difference persisted. To determine the mechanism by which IGFBP‐2 functions, the interaction between IGFBP‐2 and receptor tyrosine phosphatase β (RPTPβ) was examined. Disruption of this interaction inhibited the ability of IGFBP‐2 to stimulate AKT activation and osteoblast differentiation. Knockdown of RPTPβ enhanced osteoblast differentiation, whereas overexpression of RPTPβ was inhibitory. Adding back IGFBP‐2 to RPTPβ‐overexpressing cells was able to rescue cell differentiation via enhancement of AKT activation. To determine the region of IGFBP‐2 that mediated this effect, an IGFBP‐2 mutant that contained substitutions of key amino acids in the heparin‐binding domain‐1 (HBD‐1) was prepared. This mutant had a major reduction in its ability to stimulate differentiation of calvarial osteoblasts from IGFBP‐2−/− mice. Addition of a synthetic peptide that contained the HBD‐1 sequence to calvarial osteoblasts from IGFBP‐2−/− mice rescued differentiation and osteocalcin expression. In summary, the results clearly demonstrate that IGFBP‐2 stimulates osteoblast differentiation and that this effect is mediated through its heparin‐binding domain‐1 interacting with RPTPβ. The results suggest that stimulation of differentiation is an important mechanism by which IGFBP‐2 regulates the acquisition of normal bone mass in mice.

The Heparin-Binding Domains of IGFBP-2 Mediate Its Inhibitory Effect on Preadipocyte Differentiation and Fat Development in Male Mice

IGF-binding protein (IGFBP)-2 overexpression confers resistance to high-fat feeding and inhibits the differentiation of preadipocytes in vitro. However, whether administration of IGFBP-2 can regulate adipogenesis in vivo and the domains that mediate this response have not been defined. IGFBP-2 contains 2 heparin-binding domains (HBD), which are localized in the linker region (HBD1) and C-terminal region (HBD2) of IGFBP-2. To determine the relative importance of these domains, we used synthetic peptides as well as mutagenesis. Both HBD1 and HBD2 peptides inhibited preadipocyte differentiation, but the HBD2 peptide was more effective. Selective substitution of charged residues in the HBD1 or HBD2 regions attenuated the ability of the full-length protein to inhibit cell differentiation, but the HBD2 mutant had the greatest reduction. To determine their activities in vivo, pegylated forms of each peptide were administered to IGFBP-2(-/-) mice for 12 weeks. Magnetic resonance imaging scanning showed that only the HBD2 peptide significantly reduced (48 ± 9%, P < .05) gain in total fat mass. Both inguinal (32 ± 7%, P < .01) and visceral fat (44 ± 7%, P < .01) were significantly decreased by HBD2 whereas HBD1 reduced only visceral fat accumulation (24 ± 5%, P < .05). The HBD2 peptide was more effective peptide in reducing triglyceride content and serum adiponectin, but only the HBD2 peptide increased serum leptin. These findings demonstrate that the HBD2 domain of IGFBP-2 is the primary region that accounts for its ability to inhibit adipogenesis and that a peptide encompassing this region has activity that is comparable with native IGFBP-2.

An essential role for the association of CD47 to SHPS-1 in skeletal remodeling

Integrin‐associated protein (IAP/CD47) has been implicated in macrophage‐macrophage fusion. To understand the actions of CD47 on skeletal remodeling, we compared Cd47−/− mice with Cd47+/+ controls. Cd47−/− mice weighed less and had decreased areal bone mineral density compared with controls. Cd47−/− femurs were shorter in length with thinner cortices and exhibited lower trabecular bone volume owing to decreased trabecular number and thickness. Histomorphometry revealed reduced bone‐formation and mineral apposition rates, accompanied by decreased osteoblast numbers. No differences in osteoclast number were observed despite a nonsignificant but 40% decrease in eroded surface/bone surface in Cd47−/− mice. In vitro, the number of functional osteoclasts formed by differentiating Cd47−/− bone marrow cells was significantly decreased compared with wild‐type cultures and was associated with a decrease in bone‐resorption capacity. Furthermore, by disrupting the CD47–SHPS‐1 association, we found that osteoclastogenesis was markedly impaired. Assays for markers of osteoclast maturation suggested that the defect was at the point of fusion and not differentiation and was associated with a lack of SHPS‐1 phosphorylation, SHP‐1 phosphatase recruitment, and subsequent dephosphorylation of non–muscle cell myosin IIA. We also demonstrated a significant decrease in osteoblastogenesis in bone marrow stromal cells derived from Cd47−/− mice. Our finding of cell‐autonomous defects in Cd47−/− osteoblast and osteoclast differentiation coupled with the pronounced skeletal phenotype of Cd47−/− mice support the conclusion that CD47 plays an important role in regulating skeletal acquisition and maintenance through its actions on both bone formation and bone resorption.

Hyperglycemia stimulates p62/PKCζ interaction, which mediates NF-κB activation, increased Nox4 expression, and inflammatory cytokine activation in vascular smooth muscle

Hyperglycemia leads to vascular smooth muscle cell (VSMC) dedifferentiation and enhances responses to IGF‐I. Prior studies showed that hyperglycemia stimulated NADPH oxidase 4 (Nox4) synthesis, and IGF‐I facilitated its recruitment to a signaling complex where it oxidized src, leading to AKT and MAPK activation. To determine the mechanism that led to these changes, we analyzed the roles of p62 (sequestrosome1) and PKCζ. Hyperglycemia induced a 4.9 ± 1.0‐fold increase in p62/PKCζ association, and disruption of PKCζ/p62 using a peptide inhibitor or p62 knockdown reduced PKCζ activation (78 ± 6%). 3‐Phosphoinoside—dependent protein kinase 1 was also recruited to the p62 complex and directly phosphorylated PKCζ, leading to its activation (3.1 ± 0.4‐fold). Subsequently, activated PKCζ phosphorylated p65 rel, which led to increased Nox4 synthesis. Studies in diabetic mice confirmed these findings (6.0 ± 0.4‐fold increase in p62/PKCζ) and their disruption of attenuated Nox4 synthesis (76 ± 9% reduction). PKCζ/p62 activation stimulated inflammatory cytokine production and enhanced IGF‐I‐stimulated VSMC proliferation. These results define the molecular mechanism by which PKCζ is activated in response to hyperglycemia and suggest that this could be a mechanism by which other stimuli such as cytokines or metabolic stress function to stimulate NF‐κB activation, thereby altering VSMC sensitivity to IGF‐I.—Xi, G., Shen, X., Wai, C., Vilas, C. K., Clemmons, D. R. Hyperglycemia stimulates p62/PKCζ interaction, which mediates NF‐κB activation, increased Nox4 expression, and inflammatory cytokine activation in vascular smooth muscle. FASEB J. 29, 4772–4782 (2015). www.fasebj.org

Blocking αVβ3 integrin ligand occupancy inhibits the progression of albuminuria in diabetic rats.

This study determined if blocking ligand occupancy of the αVβ3 integrin could inhibit the pathophysiologic changes that occur in the early stages of diabetic nephropathy (DN). Diabetic rats were treated with either vehicle or a monoclonal antibody that binds the β3 subunit of the αVβ3 integrin. After 4 weeks of diabetes the urinary albumin to creatinine ratio (UACR) increased in both diabetic animals that subsequently received vehicle and in the animals that subsequently received the anti-β3 antibody compared with control nondiabetic rats. After 8 weeks of treatment the UACR continued to rise in the vehicle-treated rats; however it returned to levels comparable to control nondiabetic rats in rats treated with the anti-β3 antibody. Treatment with the antibody prevented the increase of several profibrotic proteins that have been implicated in the development of DN. Diabetes was associated with an increase in phosphorylation of the β3 subunit in kidney homogenates from diabetic animals, but this was prevented by the antibody treatment. This study demonstrates that, when administered after establishment of early pathophysiologic changes in renal function, the anti-β3 antibody reversed the effects of diabetes normalizing albuminuria and profibrotic proteins in the kidney to the levels observed in nondiabetic control animals.

The Coordinate Cellular Response to Insulin-like Growth Factor-I (IGF-I) and Insulin-like Growth Factor-binding Protein-2 (IGFBP-2) Is Regulated through Vimentin Binding to Receptor Tyrosine Phosphatase β (RPTPβ)

Background: IGFBP-2 binding to RPTPβ is required for IGF-I-stimulated AKT activation. Results: IGF-I stimulates PKCζ recruitment and serine phosphorylation of vimentin leading to vimentin/RPTPβ association, RPTPβ polymerization, and enhanced AKT activation. Conclusion: Vimentin phosphorylation stimulates vimentin/RPTPβ association, which mediates RPTPβ polymerization in response to IGF-I and IGFBP-2. Significance: This study demonstrates how these two receptor systems collaborate to obtain optimal IGF-I signal transduction. Insulin-like growth factor-binding protein-2 (IGFBP-2) functions coordinately with IGF-I to stimulate cellular proliferation and differentiation. IGFBP-2 binds to receptor tyrosine phosphatase β (RPTPβ), and this binding in conjunction with IGF-I receptor stimulation induces RPTPβ polymerization leading to phosphatase and tensin homolog inactivation, AKT stimulation, and enhanced cell proliferation. To determine the mechanism by which RPTPβ polymerization is regulated, we analyzed the protein(s) that associated with RPTPβ in response to IGF-I and IGFBP-2 in vascular smooth muscle cells. Proteomic experiments revealed that IGF-I stimulated the intermediate filament protein vimentin to bind to RPTPβ, and knockdown of vimentin resulted in failure of IGFBP-2 and IGF-I to stimulate RPTPβ polymerization. Knockdown of IGFBP-2 or inhibition of IGF-IR tyrosine kinase disrupted vimentin/RPTPβ association. Vimentin binding to RPTPβ was mediated through vimentin serine phosphorylation. The serine threonine kinase PKCζ was recruited to vimentin in response to IGF-I and inhibition of PKCζ activation blocked these signaling events. A cell-permeable peptide that contained the vimentin phosphorylation site disrupted vimentin/RPTPβ association, and IGF-I stimulated RPTPβ polymerization and AKT activation. Integrin-linked kinase recruited PKCζ to SHPS-1-associated vimentin in response to IGF-I and inhibition of integrin-linked kinase/PKCζ association reduced vimentin serine phosphorylation. PKCζ stimulation of vimentin phosphorylation required high glucose and vimentin/RPTPβ-association occurred only during hyperglycemia. Disruption of vimetin/RPTPβ in diabetic mice inhibited RPTPβ polymerization, vimentin serine phosphorylation, and AKT activation in response to IGF-I, whereas nondiabetic mice showed no difference. The induction of vimentin phosphorylation is important for IGFBP-2-mediated enhancement of IGF-I-stimulated proliferation during hyperglycemia, and it coordinates signaling between these two receptor-linked signaling systems.