Xiangdong Wang

Growth Hormone Receptor Is a Target for Presenilin-dependent γ-Secretase Cleavage

Growth hormone receptor (GHR) is a cytokine receptor superfamily member that binds growth hormone (GH) via its extracellular domain and signals via interaction of its cytoplasmic domain with JAK2 and other signaling molecules. GHR is a target for inducible metalloprotease-mediated cleavage in its perimembranous extracellular domain, a process that liberates the extracellular domain as the soluble GH-binding protein and leaves behind a cell-associated GHR remnant protein containing the transmembrane and cytoplasmic domains. GHR metalloproteolysis can be catalyzed by tumor necrosis factor-α-converting enzyme (ADAM-17) and is associated with down-modulation of GH signaling. We now study the fate of the GHR remnant protein. By anti-GHR cytoplasmic domain immunoblotting, we observed that the remnant induced in response to phorbol ester or platelet-derived growth factor has a reliable pattern of appearance and disappearance in both mouse preadipocytes endogenously expressing GHR and transfected fibroblasts expressing rabbit GHR. Lactacystin, a specific proteasome inhibitor, did not appreciably change the time course of remnant appearance or clearance but allowed detection of the GHR stub, a receptor fragment slightly smaller than the remnant but containing the C terminus of the remnant (receptor cytoplasmic domain). In contrast, MG132, another (less specific) proteasome inhibitor, strongly inhibited remnant clearance and prevented stub appearance. Inhibitors of γ-secretase, an aspartyl protease, also prevented the appearance of the stub, even in the presence of lactacystin, and concomitantly inhibited remnant clearance in the same fashion as MG132. In addition, mouse embryonic fibroblasts derived from presenilin 1 and 2 (PS1/2) knockouts recapitulated the γ-secretase inhibitor studies, as compared with their littermate controls (PS1/2 wild type). Confocal microscopy indicated that the GHR cytoplasmic domain became localized to the nucleus in a fashion dependent on PS1/2 activity. These data indicate that the GHR is subject to sequential proteolysis by metalloprotease and γ-secretase activities and may suggest GH-independent roles for the GHR.

Endotoxin-induced proteolytic reduction in hepatic growth hormone (GH) receptor: a novel mechanism for GH insensitivity.

GH is an important anabolic hormone. We previously demonstrated in cell culture that the cell surface GH receptor (GHR) is susceptible to inducible metalloproteolytic cleavage that yields the shed receptor extracellular domain (called GH binding protein) and renders the cells desensitized to subsequent GH stimulation. Sepsis and inflammatory states are associated with hepatic desensitization to GH, although disparate mechanisms have been postulated in various animal models. Using C3H/HeJ mice, we now demonstrate that administration of lipopolysaccharide (LPS) causes marked hepatic desensitization to GH, assessed by monitoring signal transducer and activator of transcription 5 tyrosine phosphorylation and nuclear accumulation and with a novel noninvasive bioluminescence imaging system to track in vivo hepatic GH signaling serially in individual mice. This endotoxin-induced desensitization was accompanied by marked loss of hepatic GHR, which was not explained by changes in GHR mRNA abundance. Furthermore, we observe that LPS causes GH-binding protein shedding of a hepatically expressed wild-type GHR but not a GHR with a mutation in the metalloprotease cleavage site. These data suggest that in this model system, LPS-induced desensitization to GH is associated with proteolytic GHR cleavage. These data are the first to demonstrate inducible in vivo GHR proteolysis and suggest this is a mechanism to regulate GH sensitivity and its anabolic effects during sepsis or inflammation.

Deletion of IGF-I receptor (IGF-IR) in primary osteoblasts reduces GH-induced STAT5 signaling.

GH promotes longitudinal growth and regulates multiple cellular functions in humans and animals. GH signals by binding to GH receptor (GHR) to activate the tyrosine kinase, Janus kinase 2 (JAK2), and downstream pathways including signal transducer and activator of transcription 5 (STAT5), thereby regulating expression of genes including IGF-I. GH exerts effects both directly and via IGF-I, which signals by activating the IGF-I receptor (IGF-IR). IGF-IR is a cell surface receptor that contains intrinsic tyrosine kinase activity within its intracellular domain. In this study, we examined the potential role of IGF-IR in facilitating GH-induced signal transduction, using mouse primary calvarial osteoblasts with Lox-P sites flanking both IGF-IR alleles. These cells respond to both GH and IGF-I and in vitro infection with an adenovirus that drives expression of Cre recombinase (Ad-Cre) dramatically reduces IGF-IR abundance without affecting the abundance of GHR, JAK2, STAT5, or ERK. Notably, infection with Ad-Cre, but not a control adenovirus, markedly inhibited acute GH-induced STAT5 activity (more than doubling the ED(50) and reducing the maximum activity by nearly 50%), while sparing GH-induced ERK activity, and markedly inhibited GH-induced transactivation of a STAT5-dependent luciferase reporter. The effect of Ad-Cre on GH signaling was specific, as platelet-derived growth factor-induced signaling was unaffected by Ad-Cre-mediated reduction of IGF-IR. Ad-Cre-mediated inhibition of GH signaling was reversed by adenoviral reexpression of IGF-IR, but not by infection with an adenovirus that drives expression of a hemagglutination-tagged somatostatin receptor, which drives expression of the unrelated somatostatin receptor, and Ad-Cre infection of nonfloxed osteoblasts did not affect GH signaling. Notably, infection with an adenovirus encoding a C-terminally truncated IGF-IR that lacks the tyrosine kinase domain partially rescued both acute GH-induced STAT5 activity and GH-induced IGF-I gene expression in cells in which endogenous IGF-IR was reduced. These data, in concert with our earlier findings that GH induces a GHR-JAK2-IGF-IR complex, suggest a novel function for IGF-IR. In addition to its role as a key IGF-I signal transducer, this receptor may directly facilitate acute GH signaling. The implications of these findings are discussed.

Involvement of CSE/ H2S in high glucose induced aberrant secretion of adipokines in 3T3-L1 adipocytes

BackgroundDeregulated secretion of adipokines contributes to subclinical systemic inflammation associated with type 2 diabetes mellitus (T2DM). However, the mechanisms underlying are not fully understood. Hydrogen sulfide (H2S), as an endogenous gasotransmitter, possesses an anti-inflammation activity. The aim of this study was to examine the possible involvement of H2S in high glucose induced adipokine secretion in 3T3-L1 adipocytes.MethodsThe expression of cystathionine-gamma-lyase (CSE), the H2S-forming enzyme, was evaluated by Western-blotting and real-time PCR. The secretion of TNF-α, MCP-1 and adiponectin was determined by radioimmunoassay and enzyme-linked immunosorbent assay (ELISA), respectively. Lentiviral empty vector and vector expressing mouse CSE were used for in vitro transduction.ResultsHigh glucose (HG) significantly decreased CSE expression at both protein and mRNA levels in mature 3T3-L1 adipocytes. In parallel, HG significantly increased secretion of MCP-1 while decreasing secretion of adiponectin, but had no effect on secretion of TNF-α. HG induced changes in MCP-1 and adiponectin secretion were partly attenuated by forced expression of CSE or sodium hydrosulfide (NaHS), a source of exogenous H2S.ConclusionHigh glucose induces aberrant secretion of adipokines in mature 3T3-L1 adipocytes, favoring inflammation. The mechanism is partly related to inhibition of CSE/ H2S system.

Growth hormone receptor targeting to lipid rafts requires extracellular subdomain 2

GH receptor (GHR) is a single membrane-spanning glycoprotein dimer that binds GH in its extracellular domain (ECD). GH activates the GHR intracellular domain (ICD)-associated tyrosine kinase, JAK2, which causes intracellular signaling. We previously found that plasma membrane (PM)-associated GHR was dramatically enriched in the lipid raft (LR) component of the membrane and that localization of GHR within PM regions may regulate GH signaling by influencing the profile of pathway activation. In this study, we examined determinants of LR localization of the GHR using a reconstitution system which lacks endogenous JAK2 and GHR. By non-detergent extraction and multistep fractionation, we found that GHR was highly enriched in the LR fraction independent of JAK2 expression. Various GHR mutants were examined in transfectants harboring JAK2. LR concentration was observed for a GHR in which the native transmembrane domain (TMD) is replaced by that of the unrelated LDL receptor and for a GHR that lacks its ICD. Thus, LR association requires neither the TMD nor the ICD. Similarly, a GHR that lacks the ECD, except for the membrane-proximal ECD stem region, was only minimally LR-concentrated. Mutants with internal stem deletions in the context of the full-length receptor were LR-concentrated similar to the wild-type. A GHR lacking ECD subdomain 1 reached the PM and was LR-concentrated, while one lacking ECD subdomain 2, also reached the PM, but was not LR-concentrated. These data suggest LR targeting resides in ECD subdomain 2, a region relatively uninvolved in GH binding.

Inhibitory GH Receptor Extracellular Domain Monoclonal Antibodies: Three-Dimensional Epitope Mapping

GH receptor (GHR) mediates the anabolic and metabolic effects of GH. We previously characterized a monoclonal antibody (anti-GHR(ext-mAb)) that reacts with subdomain 2 of the rabbit GHR extracellular domain (ECD) and is a conformation-specific inhibitor of GH signaling in cells bearing rabbit or human GHR. Notably, this antibody has little effect on GH binding and also inhibits inducible metalloproteolysis of the GHR that occurs in the perimembranous ECD stem region. In the current study, we demonstrate that anti-GHR(ext-mAb) inhibits GH-dependent cellular proliferation and also inhibits hepatic GH signaling in vivo in mice that adenovirally express rabbit GHR, as assessed with our noninvasive bioluminescence hepatic signaling assay. A separate monoclonal antibody (anti-GHR(mAb 18.24)) is a sister clone of anti-GHR(ext-mAb). Here, we demonstrate that anti-GHR(mAb 18.24) also inhibits rabbit and human GHR signaling and inducible receptor proteolysis. Further, we use a random PCR-generated mutagenic expression system to map the three-dimensional epitopes in the rabbit GHR ECD for both anti-GHR(ext-mAb) and anti-GHR(mAb 18.24). We find that each of the two antibodies has similar, but nonidentical, discontinuous epitopes that include regions of subdomain 2 encompassing the dimerization interface. These results have fundamental implications for understanding the role of the dimerization interface and subdomain 2 in GHR activation and regulated GHR metalloproteolysis and may inform development of therapeutics that target GHR.

Interruption of Growth Hormone Signaling via SHC and ERK in 3T3-F442A Preadipocytes upon Knockdown of Insulin Receptor Substrate-1

Insulin receptor substrate-1 (IRS-1) is a docking protein tyrosine phosphorylated in response to insulin, IGF-1, GH, and other cytokines. IRS-1 has an N-terminal plekstrin homology domain (which facilitates membrane localization), a phosphotyrosine-binding domain [which associates with tyrosine-phosphorylated insulin receptor or IGF-1 receptor (IGF-1R)], and tyrosine residues that, when phosphorylated, bind signaling molecules. The role of IRS-1 in GH signaling is uncertain. We previously reported that IRS-1 and Janus kinase 2 associate independently of tyrosine phosphorylation via IRS-1s N terminus and that IRS-1 reconstitution greatly enhances GH-induced ERK, but not STAT5, activation. We now use GH-responsive 3T3-F442A preadipocytes to study the influence of IRS-1 on GH action. We stably transfected cells with vector only (Control) or a vector encoding IRS-1 short hairpin RNA [knockdown (KD)] and compared representative clones. Immunoblotting confirmed more than 80% knockdown of IRS-1 in KD cells. GH caused characteristic Janus kinase 2 and STAT5 activation in both Control and KD cells, but ERK activation was dramatically reduced in KD cells in GH time course and dose-response experiments. Notably, GH-induced Src homology collagen (SHC) activation and SHC-Grb2 association in KD cells were also markedly diminished compared with Control cells. Subcellular fractionation revealed that IRS-1 in Control cells was largely cytosolic, but the component isolated with plasma membranes was highly enriched in lipid raft membranes (LR). In KD cells, GH-induced ERK activation in the LR fraction was particularly diminished compared with Control cells. These data suggest that LR-enriched IRS-1 contributes substantially to GH-induced ERK activation in LR in 3T3-F442A fibroblasts. Furthermore, our results are consistent with IRS-1 residing upstream of SHC in the GH-induced ERK-signaling pathway.

The Orphan Nuclear Receptor NR4A1 Protects Pancreatic β-Cells from Endoplasmic Reticulum (ER) Stress-mediated Apoptosis

Background: It is not known whether NR4A1 plays a role in ER stress-induced β-cell apoptosis. Results: NR4A1 expression in β-cells or islets correlated positively with Survivin expression and negatively with CHOP expression and apoptosis rate upon treatment with ER stress inducers. Conclusion: NR4A1 protects β-cells from ER stress-induced apoptosis by up-regulating Survivin and down-regulating CHOP expression. Significance: Our findings provide clues to prevent diabetes. The role of NR4A1 in apoptosis is controversial. Pancreatic β-cells often face endoplasmic reticulum (ER) stress under adverse conditions such as high free fatty acid (FFA) concentrations and sustained hyperglycemia. Severe ER stress results in β-cell apoptosis. The aim of this study was to analyze the role of NR4A1 in ER stress-mediated β-cell apoptosis and to characterize the related mechanisms. We confirmed that upon treatment with the ER stress inducers thapsigargin (TG) or palmitic acid (PA), the mRNA and protein levels of NR4A1 rapidly increased in both MIN6 cells and mouse islets. NR4A1 overexpression in MIN6 cells conferred resistance to cell loss induced by TG or PA, as assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, and TUNEL assays indicated that NR4A1 overexpression also protected against ER stress-induced apoptosis. This conclusion was further confirmed by experiments exploiting siRNA to knockdown NR4A1 expression in MIN6 cells or exploiting NR4A1 knock-out mice. NR4A1 overexpression in MIN6 cells reduced C/EBP homologous protein (CHOP) expression and Caspase3 activation induced by TG or PA. NR4A1 overexpression in MIN6 cells or mouse islets resulted in Survivin up-regulation. A critical regulatory element was identified in Survivin promoter (−1872 bp to −1866 bp) with a putative NR4A1 binding site; ChIP assays demonstrated that NR4A1 physically associates with the Survivin promoter. In conclusion, NR4A1 protects pancreatic β-cells against ER stress-mediated apoptosis by up-regulating Survivin expression and down-regulating CHOP expression, which we termed as “positive and negative regulation.”

Small islets transplantation superiority to large ones: implications from islet microcirculation and revascularization.

Pancreatic islet transplantation is a promising therapy to regain glycemic control in diabetic patients. The selection of ideal grafts is the basis to guarantee short-term effectivity and longevity of the transplanted islets. Contradictory to the traditional notion, recent findings implied the superiority of small islets for better transplantation outcomes rather than the large and intact ones. However, the mechanisms remain to be elucidated. Recent evidences emphasized the major impact of microcirculation on islet β-cell mass and function. And potentials in islet graft revascularization are crucial for their survival and preserved function in the recipient. In this study, we verified the distinct histological phenotype and functionality of small islets versus large ones both in vitro and in vivo. With efforts to exploring the differences in microcirculation and revascularization of islet grafts, we further evaluated local expressions of angiotensin and vascular endothelial growth factor A (VEGF-A) at different levels. Our findings reveal that, apart from the higher density of insulin-producing β-cells, small islets express less angiotensin and more angiotrophic VEGF-A. We therefore hypothesized a logical explanation of the small islet superiority for transplantation outcome from the aspects of facilitated microcirculation and revascularization intrinsically in small islets.

Twist 1 regulates the expression of PPARγ during hormone-induced 3T3-L1 preadipocyte differentiation: a possible role in obesity and associated diseases

BackgroundTwist 1 is highly expressed in adipose tissue and has been associated with obesity and related disorders. However, the molecular function of Twist 1 in adipose tissue is unclear. Twist 1 has been implicated in cell lineage determination and differentiation. Therefore, we investigated both the role of Twist 1 in adipocyte precursor mobilization and the relationship of Twist 1 with other molecular determinants of adipocyte differentiation.MethodsWe examined Twist 1 mRNA and protein expression in subcutaneous adipose tissues from diet-induced obese C57/BL6 mice and Wistar rats and in obese patients undergoing liposuction or adipose transplant surgeries. Twist 1 expression was measured on days 0, 2, 4, 8, and 12 of 3T3-L1 differentiation in vitro. The role of Twist 1 in adipogenesis was explored using retroviral interference of Twist 1 expression. Adipokine secretion was evaluated using a RayBio® Biotin Label-based Adipokine Array.ResultsTwist 1 mRNA and protein levels were reduced in diet-induced obese mice and rats and in obese humans. Twist 1 was upregulated during 3T3-L1 preadipocyte differentiation in vitro, beginning from the fourth day of differentiation induction. Retroviral interference of Twist 1 expression did not significantly impair lipid formation; however, retroviral interference induced PPARγ mRNA and protein expression on day 4 of differentiation induction. Adipokine array analyses revealed increased secretion of CXCR4 (19.55-fold), VEGFR1 (92.13-fold), L-21 R (63.55-fold), and IL-12 R beta 1 (59.66-fold) and decreased secretion of VEGFR3 (0.01-fold), TSLP R (0.071-fold), MIP-1 gamma (0.069-fold), TNF RI/TNFRSF1A (0.09-fold), and MFG-E8 (0.06-fold).ConclusionsTwist 1 is a regulator of adipocyte gene expression although it is not likely to regulate differentiation. We identified PPARγ as a potential target of Twist 1 and found variation in the secretion of multiple adipokines, which might indicate a prospective mechanism linking Twist 1 expression with obesity or associated diseases.