Interpreting growth hormone and IGF-I results using modern assays and reference ranges for the monitoring of treatment effectiveness in acromegaly.

Standard treatment for acromegaly focuses on the achievement of target absolute levels of growth hormone (GH) and insulin-like growth factor (IGF-I). The appropriateness of these targets when measured using modern assay methods is not well defined. This paper reviews biochemical status assessed using methods available at the time and associated clinical outcomes. GH measurements were shown to provide an indication of changes in tumor size, and failure of GH suppression after glucose stimulation is associated with tumor recurrence. IGF-I levels were more closely associated with changes in symptoms and signs. Reduced GH and IGF-I concentrations were shown to be associated with increased longevity, although the degree of increase has only been analyzed for GH. Lowering of GH and IGF-I has consistently been associated with improved outcomes; however, absolute levels reported in previous studies were based on results from methods and reference ranges that are now obsolete. Applying previously described absolute thresholds as targets (e.g. "normal" IGF-I level) when using current methods is best applied to those with active acromegaly symptoms who could benefit from further lowering of biochemical markers. In asymptomatic individuals with mild IGF-I or GH elevations, targeting biochemical "normalization" would result in the need for combination pharmacotherapy in many patients without proven benefit. Measurement of both GH and IGF-I remains an essential component of diagnosis and monitoring the effectiveness of treatment in acromegaly; however, treatment goals based only on previously identified absolute thresholds are not appropriate without taking into account the assay and reference ranges being employed. Treatment goals should be individualized considering biochemical improvement from an untreated baseline, symptoms of disease, risks, burdens and costs of complex treatment regimens, comorbidities, and quality of life.

Development of a Novel Algorithm to Identify People with High Likelihood of Adult Growth Hormone Deficiency in a US Healthcare Claims Database.

Objective: Adult growth hormone deficiency (AGHD) is an underdiagnosed disease associated with increased morbidity and mortality. Identifying people who may benefit from growth hormone (GH) therapy can be challenging, as many AGHD symptoms resemble those of aging. We developed an algorithm to potentially help providers stratify people by their likelihood of having AGHD. Design: The algorithm was developed with, and applied to, data in the anonymized Truven Health MarketScan® claims database. Patients. A total of 135 million adults in the US aged ≥18 years with ≥6 months of data in the Truven database. Measurements. Proportion of people with high, moderate, or low likelihood of having AGHD, and differences in demographic and clinical characteristics among these groups. Results: Overall, 0.5%, 6.0%, and 93.6% of people were categorized into groups with high, moderate, or low likelihood of having AGHD, respectively. The proportions of females were 59.3%, 71.6%, and 50.4%, respectively. People in the high- and moderate-likelihood groups tended to be older than those in the low-likelihood group, with 58.3%, 49.0%, and 37.6% aged >50 years, respectively. Only 2.2% of people in the high-likelihood group received GH therapy as adults. The high-likelihood group had a higher incidence of comorbidities than the low-likelihood group, notably malignant neoplastic disease (standardized difference -0.42), malignant breast tumor (-0.27), hyperlipidemia (-0.26), hypertensive disorder (-0.25), osteoarthritis (-0.23), and heart disease (-0.22). Conclusions: This algorithm may represent a cost-effective approach to improve AGHD detection rates by identifying appropriate patients for further diagnostic testing and potential GH replacement treatment.

Pituitary Neoplasm Nomenclature Workshop: Does Adenoma Stand the Test of Time?

The WHO Classification of Endocrine Tumours designates pituitary neoplasms as adenomas. A proposed nomenclature change to pituitary neuroendocrine tumors (PitNETs) has been met with concern by some stakeholder groups. The Pituitary Society coordinated the Pituitary Neoplasm Nomenclature (PANOMEN) workshop to address the topic. Experts in pituitary developmental biology, pathology, neurosurgery, endocrinology, and oncology, including representatives nominated by the Endocrine Society, European Society of Endocrinology, European Neuroendocrine Association, Growth Hormone Research Society, and International Society of Pituitary Surgeons. Clinical epidemiology, disease phenotype, management, and prognosis of pituitary adenomas differ from that of most NETs. The vast majority of pituitary adenomas are benign and do not adversely impact life expectancy. A nomenclature change to PitNET does not address the main challenge of prognostic prediction, assigns an uncertain malignancy designation to benign pituitary adenomas, and may adversely affect patients. Due to pandemic restrictions, the workshop was conducted virtually, with audiovisual lectures and written précis on each topic provided to all participants. Feedback was collated and summarized by Content Chairs and discussed during a virtual writing meeting moderated by Session Chairs, which yielded an evidence-based draft document sent to all participants for review and approval. There is not yet a case for adopting the PitNET nomenclature. The PANOMEN Workshop recommends that the term adenoma be retained and that the topic be revisited as new evidence on pituitary neoplasm biology emerges.

Estrogen Stimulation of Pleiotrophin Enhances Osteoblast Differentiation and Maintains Bone Mass in IGFBP-2 Null Mice.

Insulin-like growth factor binding protein-2 (IGFBP-2) stimulates osteoblast differentiation but only male Igfbp2 null mice have a skeletal phenotype. The trophic actions of IGFBP-2 in bone are mediated through its binding to receptor tyrosine phosphatase beta (RPTPß). Another important ligand for RPTPß is pleiotrophin (PTN), which also stimulates osteoblast differentiation. We determined the change in PTN and RPTPß in Igfbp2-/- mice. Analysis of whole bone mRNA in wild-type and knockout mice revealed increased expression of Ptn. Rptpß increased in gene-deleted animals with females having greater expression than males. Knockdown of PTN expression in osteoblasts in vitro inhibited differentiation, and addition of PTN to the incubation medium rescued the response. Estradiol stimulated PTN secretion and PTN knockdown blocked estradiol-stimulated differentiation. PTN addition to IGFBP-2 silenced osteoblast stimulated differentiation, and an anti-fibronectin-3 antibody, which inhibits PTN binding to RPTPß, inhibited this response. Estrogen stimulated PTN secretion and downstream signaling in the IGFBP-2 silenced osteoblasts and these effects were inhibited with anti-fibronectin-3. Administration of estrogen to wild-type and Igfbp2-/- male mice stimulated an increase in both areal bone mineral density and trabecular bone volume fraction but the increase was significantly greater in the Igfbp2-/- animals. Estrogen also stimulated RPTPß expression in the null mice. We conclude that loss of IGFBP-2 expression is accompanied by upregulation of PTN and RPTPß expression in osteoblasts, that the degree of increase is greater in females due to estrogen secretion, and that this compensatory change may account for some component of the maintenance of normal bone mass in female mice.

THYROID DYSFUNCTION, RECOVERY, AND PROGNOSIS IN MELANOMA PATIENTS TREATED WITH IMMUNE CHECKPOINT INHIBITORS: A RETROSPECTIVE REVIEW.

Objective: To describe thyroid dysfunction, factors associated with thyroid recovery, and survival in melanoma patients treated with immune checkpoint inhibitors that developed thyroid immune-related adverse events (irAEs). Methods: This was a retrospective study in a tertiary center from 2010-2017. We reviewed the charts of patients with melanoma that developed thyroid dysfunction after checkpoint inhibitor therapy. Cases with thyroid irAEs were grouped by recovery of thyroid function at 1 year. We collected a timeline of thyroid function tests, medication exposure, and survival and compared variables between the groups. We studied survival in comparison to a matched group without thyroid dysfunction. Results: A total of 186 melanoma patients received checkpoint inhibitors, and 17 (9%) had thyroid irAEs. Median time to abnormal thyroid-stimulating hormone was 38 days and followed a pattern of thyroiditis. Seven of 17 had thyroid recovery. In the no-recovery group, free thyroxine (T4) was often above 2 ng/dL (5/10 in no recovery, 0/7 in recovery; P = .04). In the recovery group, irAE grade was significantly lower, with 7/7 grade 1 (P = .004). Exposure to glucocorticoids was associated with recovery (3/10 in no recovery, 6/7 in recovery; P = .049). There was no difference in overall survival between the thyroid dysfunction group and controls, or between those that received glucocorticoids or not. Conclusion: Certain aspects of thyroid irAEs may correlate with thyroid recovery, including grade 1 thyroid irAEs, exposure to glucocorticoids, and peak free T4 levels less than 2 ng/dL. Thyroid irAEs did not appear to be associated with change in survival nor did exposure to glucocorticoids. Abbreviations: ASCO = American Society of Clinical Oncology; CTLA-4 = cytotoxic T-lymphocyte-associated protein 4; irAE = immune-related adverse event; PD-1 = programmed cell death protein 1; T4 = thyroxine; TSH = thyroid-stimulating hormone.

Hyperglycemia induces vascular smooth muscle cell dedifferentiation by suppressing insulin receptor substrate-1-mediated p53/KLF4 complex stabilization.

Hyperglycemia and insulin resistance accelerate atherosclerosis by an unclear mechanism. The two factors down-regulate insulin receptor substrate-1 (IRS-1), an intermediary of the insulin/IGF-I signaling system. We previously reported that IRS-1 down-regulation leads to vascular smooth muscle cell (VSMC) dedifferentiation and that IRS-1 deletion from VSMCs in normoglycemic mice replicates this response. However, we did not determine IRS-1's role in mediating differentiation. Here, we sought to define the mechanism by which IRS-1 maintains VSMC differentiation. High glucose or IRS-1 knockdown decreased p53 levels by enhancing MDM2 proto-oncogene (MDM2)-mediated ubiquitination, resulting in decreased binding of p53 to Krüppel-like factor 4 (KLF4). Exposure to nutlin-3, which dissociates MDM2/p53, decreased p53 ubiquitination and enhanced the p53/KLF4 association and differentiation marker protein expression. IRS-1 overexpression in high glucose inhibited the MDM2/p53 association, leading to increased p53 and p53/KLF4 levels, thereby increasing differentiation. Nutlin-3 treatment of diabetic or Irs1-/- mice enhanced p53/KLF4 and the expression of p21, smooth muscle protein 22 (SM22), and myocardin and inhibited aortic VSMC proliferation. Injecting normoglycemic mice with a peptide disrupting the IRS-1/p53 association reduced p53, p53/KLF4, and differentiation. Analyzing atherosclerotic lesions in hypercholesterolemic, diabetic pigs, we found that p53, IRS-1, SM22, myocardin, and KLF4/p53 levels are significantly decreased compared with their expression in nondiabetic pigs. We conclude that IRS-1 is critical for maintaining VSMC differentiation. Hyperglycemia- or insulin resistance-induced IRS-1 down-regulation decreases the p53/KLF4 association and enhances dedifferentiation and proliferation. Our results suggest that enhancing IRS-1-dependent p53 stabilization could attenuate the progression of atherosclerotic lesions in hyperglycemia and insulin-resistance states.

A peptide containing the receptor binding site of insulin-like growth factor binding protein-2 enhances bone mass in ovariectomized rats.

Male Igfbp2-/- mice have a significant reduction in bone mass and administration of a peptide that contains the insulin-like growth factor binding protein-2(IGFBP-2) receptor-binding domain stimulates bone formation in these animals. Female Igfbp2-/- mice do not have this phenotype but following ovariectomy (OVX) lose more bone than OVX wild-type mice. This suggests that in the absence of estrogen, IGFBP-2 is required to maintain bone mass. Therefore these studies were undertaken to determine if this peptide could stimulate bone acquisition in OVX rats. OVX rats were divided into seven treatment groups: sham animals, OVX animals, OVX animals receiving a control scrambled peptide, or one of three doses of the active peptide termed PEG-HBD-1 (0.7, 2, and 6 mg·kg-1) and an OVX group receiving parathyroid hormone (PTH) (50 µg·kg-1 per day). The peptides were administered for 8 weeks. DXA revealed a significant reduction in femoral and tibial areal bone mineral density (aBMD) after OVX, whereas treatment with the high-dose peptide increased aBMD by 6.2% ± 2.4% (P < 0.01) compared to control peptide; similar to the increase noted with PTH (5.6% ± 3.0%, P < 0.01). Similar increases were noted with two lower doses of the peptide (3.8% ± 1.5%, P < 0.05 for low dose; 3.1% ± 1.6%, P = 0.07 for middle dose). Micro CT showed that the OVX control peptide animals had reductions of 41% and 64% in femoral trabecular BV/TV and trabecular number, respectively. All three doses of the peptide increased bone volume/total volume (BV/TV) significantly, while the low and middle doses increased trabecular number. Cortical BV/TV and thickness at the midshaft increased significantly with each dose of peptide (18.9% ± 9.8%, P < 0.01 and 14.2% ± 7.9%, P < 0.01 for low dose; 23.7% ± 10.7%, P < 0.001 and 15.8% ± 6.1%, P < 0.001 for middle dose; 19.0% ± 6.9%, P < 0.01 and 16.2% ± 9.7%, P < 0.001 for high dose) and with PTH (25.8% ± 9.2%, P < 0.001 and 19.4% ± 8.8%, P < 0.001). Histomorphometry showed that the lowest dose of peptide stimulated BV/TV, trabecular thickness, mineral apposition rate (MAR), bone formation rate/bone surface (BFR/BS), number of osteoblasts/bone perimeter (N.ob/B.pm), and decreased osteoclast surface/bone perimeter (Oc.S/B.Pm). The highest dose stimulated each of these parameters except MAR and BFR/BS. Thus, the heparin-binding domain receptor region of IGFBP-2 accounts for its anabolic activity in bone. Importantly, this peptide enhances bone mass in estrogen-deficient animals.

A Consensus Statement on acromegaly therapeutic outcomes.

The 11th Acromegaly Consensus Conference in April 2017 was convened to update recommendations on therapeutic outcomes for patients with acromegaly. Consensus guidelines on the medical management of acromegaly were last published in 2014; since then, new pharmacological agents have been developed and new approaches to treatment sequencing have been considered. Thirty-seven experts in the management of patients with acromegaly reviewed the current literature and assessed changes in drug approvals, clinical practice standards and clinical opinion. They considered current treatment outcome goals with a focus on the impact of current and emerging somatostatin receptor ligands, growth hormone receptor antagonists and dopamine agonists on biochemical, clinical, tumour mass and surgical outcomes. The participants discussed factors that would determine pharmacological choices as well as the proposed place of each agent in the guidelines. We present consensus recommendations highlighting how acromegaly management could be optimized in clinical practice.

Role of IGF-binding proteins in regulating IGF responses to changes in metabolism.

The IGF-binding protein family contains six members that share significant structural homology. Their principal function is to regulate the actions of IGF1 and IGF2. These proteins are present in plasma and extracellular fluids and regulate access of both IGF1 and II to the type I IGF receptor. Additionally, they have functions that are independent of their ability to bind IGFs. Each protein is regulated independently of IGF1 and IGF2, and this provides an important mechanism by which other hormones and physiologic variables can regulate IGF actions indirectly. Several members of the family are sensitive to changes in intermediary metabolism. Specifically the presence of obesity/insulin resistance can significantly alter the expression of these proteins. Similarly changes in nutrition or catabolism can alter their synthesis and degradation. Multiple hormones such as glucocorticoids, androgens, estrogen and insulin regulate IGFBP synthesis and bioavailability. In addition to their ability to regulate IGF access to receptors these proteins can bind to distinct cell surface proteins or proteins in extracellular matrix and several cellular functions are influenced by these interactions. IGFBPs can be transported intracellularly and interact with nuclear proteins to alter cellular physiology. In pathophysiologic states, there is significant dysregulation between the changes in IGFBP synthesis and bioavailability and changes in IGF1 and IGF2. These discordant changes can lead to marked alterations in IGF action. Although binding protein physiology and pathophysiology are complex, experimental results have provided an important avenue for understanding how IGF actions are regulated in a variety of physiologic and pathophysiologic conditions.

IRS-1 Functions as a Molecular Scaffold to Coordinate IGF-I/IGFBP-2 Signaling During Osteoblast Differentiation.

Insulin like growth factor I (IGF-I) and insulin like growth factor binding protein-2 (IGFBP-2) function coordinately to stimulate AKT and osteoblast differentiation. IGFBP-2 binding to receptor protein tyrosine phosphatase ß (RPTPß) stimulates polymerization and inactivation of phosphatase activity. Because phosphatase and tensin homolog (PTEN) is the primary target of RPTPß, this leads to enhanced PTEN tyrosine phosphorylation and inactivation. However RPTPß inactivation also requires IGF-I receptor activation. The current studies were undertaken to determine the mechanism by which IGF-I mediates changes in RPTPß function in osteoblasts. IGFBP-2/IGF-I stimulated vimentin binding to RPTPß and this was required for RPTPß polymerization. Vimentin serine phosphorylation mediated its binding to RPTPß and PKCζ was identified as the kinase that phosphorylated vimentin. To determine the mechanism underlying IGF-I stimulation of PKCζ-mediated vimentin phosphorylation, we focused on insulin receptor substrate-1 (IRS-1). IGF-I stimulated IRS-1 phosphorylation and recruitment of PKCζ and vimentin to phospho-IRS-1. IRS-1 immunoprecipitates containing PKCζ and vimentin were used to confirm that activated PKCζ directly phosphorylated vimentin. PKCζ does not contain a SH-2 domain that is required to bind to phospho-IRS-1. To determine the mechanism of PKCζ recruitment we analyzed the role of p62 (a PKCζ binding protein) that contains a SH2 domain. Exposure to differentiation medium plus IGF-I stimulated PKCζ/p62 association. Subsequent analysis showed the p62/PKCζ complex was co-recruited to IRS-1. Peptides that disrupted p62/PKCζ or p62/IRS-1 inhibited IGF-I/IGFBP-2 stimulated PKCζ activation, vimentin phosphorylation, PTEN tyrosine phosphorylation, AKT activation, and osteoblast differentiation. The importance of these signaling events for differentiation was confirmed in primary mouse calvarial osteoblasts. These results demonstrate the cooperative interaction between RPTPß and the IGF-I receptor leading to a coordinated series of signaling events that are required for osteoblast differentiation. Our findings emphasize the important role IRS-1 plays in modulating these signaling events and confirm its essential role in facilitating osteoblast differentiation. © 2016 American Society for Bone and Mineral Research.

Igfbp2 Deletion in Ovariectomized Mice Enhances Energy Expenditure but Accelerates Bone Loss.

Previously, we reported sexually dimorphic bone mass and body composition phenotypes in Igfbp2(-/-) mice (-/-), where male mice exhibited decreased bone and increased fat mass, whereas female mice displayed increased bone but no changes in fat mass. To investigate the interaction between IGF-binding protein (IGFBP)-2 and estrogen, we subjected Igfbp2 -/- and +/+ female mice to ovariectomy (OVX) or sham surgery at 8 weeks of age. At 20 weeks of age, mice underwent metabolic cage analysis and insulin tolerance tests before killing. At harvest, femurs were collected for microcomputed tomography, serum for protein levels, brown adipose tissue (BAT) and inguinal white adipose tissue (IWAT) adipose depots for histology, gene expression, and mitochondrial respiration analysis of whole tissue. In +/+ mice, serum IGFBP-2 dropped 30% with OVX. In the absence of IGFBP-2, OVX had no effect on preformed BAT; however, there was significant "browning" of the IWAT depot coinciding with less weight gain, increased insulin sensitivity, lower intraabdominal fat, and increased bone loss due to higher resorption and lower formation. Likewise, after OVX, energy expenditure, physical activity and BAT mitochondrial respiration were decreased less in the OVX-/- compared with OVX+/+. Mitochondrial respiration of IWAT was reduced in OVX+/+ yet remained unchanged in OVX-/- mice. These changes were associated with significant increases in Fgf21 and Foxc2 expression, 2 proteins known for their insulin sensitizing and browning of WAT effects. We conclude that estrogen deficiency has a profound effect on body and bone composition in the absence of IGFBP-2 and may be related to changes in fibroblast growth factor 21.

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 ß (RPTPß) was examined. Disruption of this interaction inhibited the ability of IGFBP-2 to stimulate AKT activation and osteoblast differentiation. Knockdown of RPTPß enhanced osteoblast differentiation, whereas overexpression of RPTPß was inhibitory. Adding back IGFBP-2 to RPTPß-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 RPTPß. The results suggest that stimulation of differentiation is an important mechanism by which IGFBP-2 regulates the acquisition of normal bone mass in mice.

Expert consensus document: A consensus on the medical treatment of acromegaly.

In March 2013, the Acromegaly Consensus Group met to revise and update guidelines for the medical treatment of acromegaly. The meeting comprised experts skilled in the medical management of acromegaly. The group considered treatment goals covering biochemical, clinical and tumour volume outcomes, and the place in guidelines of somatostatin receptor ligands, growth hormone receptor antagonists and dopamine agonists, and alternative modalities for treatment including combination therapy and novel treatments. This document represents the conclusions of the workshop consensus.

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.

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 (IGF) binding protein 2 functions coordinately with receptor protein tyrosine phosphatase ß and the IGF-I receptor to regulate IGF-I-stimulated signaling.

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 ß (RPTPß) in regulating IGF-I signaling and cellular proliferation. IGFBP-2 bound RPTPß, 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 RPTPß 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 RPTPß 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.

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.

IGF-I stimulates cooperative interaction between the IGF-I receptor and CSK homologous kinase that regulates SHPS-1 phosphorylation in vascular smooth muscle cells.

IGF-I plays an important role in smooth muscle cell proliferation and migration. In vascular smooth muscle cells cultured in 25 mm glucose, IGF-I stimulated a significant increase in Src homology 2 domain containing protein tyrosine phosphatase substrate-1 (SHPS-1) phosphorylation compared with 5 mm glucose and this increase was required for smooth muscle cell proliferation. A proteome-wide screen revealed that carboxyl-terminal SRC kinase homologous kinase (CTK) bound directly to phosphotyrosines in the SHPS-1 cytoplasmic domain. Because the kinase(s) that phosphorylates these tyrosines in response to IGF-I is unknown, we determined the roles of IGF-I receptor (IGF-IR) and CTK in mediating SHPS-1 phosphorylation. After IGF-I stimulation, CTK was recruited to IGF-IR and subsequently to phospho-SHPS-1. Expression of an IGF-IR mutant that eliminated CTK binding reduced CTK transfer to SHPS-1, SHPS-1 phosphorylation, and cell proliferation. IGF-IR phosphorylated SHPS-1, which provided a binding site for CTK. CTK recruitment to SHPS-1 resulted in a further enhancement of SHPS-1 phosphorylation. CTK knockdown also impaired IGF-I-stimulated SHPS-1 phosphorylation and downstream signaling. Analysis of specific tyrosines showed that mutation of tyrosines 428/452 in SHPS-1 to phenylalanine reduced SHPS-1 phosphorylation but allowed CTK binding. In contrast, the mutation of tyrosines 469/495 inhibited IGF-IR-mediated the phosphorylation of SHPS-1 and CTK binding, suggesting that IGF-IR phosphorylated Y469/495, allowing CTK binding, and that CTK subsequently phosphorylated Y428/452. Based on the above findings, we conclude that after IGF-I stimulation, CTK is recruited to IGF-IR and its recruitment facilitates CTK's subsequent association with phospho-SHPS-1. This results in the enhanced CTK transfer to SHPS-1, and the two kinases then fully phosphorylate SHPS-1, which is necessary for IGF-I stimulated cellular proliferation.

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.

Suppression of AMPK activation via S485 phosphorylation by IGF-I during hyperglycemia is mediated by AKT activation in vascular smooth muscle cells.

As a metabolic sensor, the serine/threonine protein kinase AMP-activated protein kinase (AMPK) promotes the adaptation of cells to signals arising from nutrients, hormones, and growth factors. The ability of IGF-I to stimulate protein synthesis is suppressed by AMPK, therefore, these studies were undertaken to determine whether IGF-I modulates AMPK activity. IGF-I dose-dependently suppressed phosphorylation of AMPK T172, and it stimulated AMPK S485 phosphorylation in vascular smooth muscle cells (VSMC). To determine whether stimulation of AMPK S485 phosphorylation was mediating this response, VSMC were transduced with a mutant AMPKα (AMPK S485A). Expression of this altered form inhibited the ability of IGF-I to suppress AMPK T172 activation, which resulted in inhibition of IGF-I-stimulated phosphorylation of P70S6 kinase. In contrast, expression of an AMPK S485D mutant resulted in constitutive suppression of AMPK activity and was associated with increased IGF-I-stimulated P70S6K phosphorylation and protein synthesis. The addition of a specific AKT inhibitor or expression of an AKT1 short hairpin RNA inhibited AMPK S485 phosphorylation, and it attenuated the IGF-I-induced decrease in AMPK T172 phosphorylation. Exposure to high glucose concentrations suppressed AMPK activity and stimulated S485 phosphorylation, and IGF-I stimulated a further increase in S485 phosphorylation and AMPK T172 suppression. We conclude that AMPK S485 phosphorylation negatively regulates AMPK activity by modulating the T172 phosphorylation response to high glucose and IGF-I. IGF-I stimulates S485 phosphorylation through AKT1. The results suggest that AMPK plays an inhibitory role in modulating IGF-I-stimulated protein synthesis and that IGF-I must down-regulate AMPK activity to induce an optimal anabolic response.