Bonnie Peng

Targeted Deletion of the Vgf Gene Indicates that the Encoded Secretory Peptide Precursor Plays a Novel Role in the Regulation of Energy Balance

To determine the function of VGF, a secreted polypeptide that is synthesized by neurons, is abundant in the hypothalamus, and is regulated in the brain by electrical activity, injury, and the circadian clock, we generated knockout mice lacking Vgf. Homozygous mutants are small, hypermetabolic, hyperactive, and infertile, with markedly reduced leptin levels and fat stores and altered hypothalamic proopiomelanocortin (POMC), neuropeptide Y (NPY), and agouti-related peptide (AGRP) expression. Furthermore, VGF mRNA synthesis is induced in the hypothalamic arcuate nuclei of fasted normal mice. VGF therefore plays a critical role in the regulation of energy homeostasis, suggesting that the study of lean VGF mutant mice may provide insight into wasting disorders and, moreover, that pharmacological antagonism of VGF action(s) might constitute the basis for treatment of obesity.

The role of prohormone convertase-2 in hypothalamic neuropeptide processing: a quantitative neuropeptidomic study

Prohormone convertase (PC) 1/3 and 2 are involved in the generation of neuropeptides from their precursors. A quantitative peptidomic approach was used to explore the role PC2 plays in the processing of hypothalamic peptides. In this approach, extracts from mice lacking PC2 activity and from wild‐type littermates were labeled with isotopic tags, combined, fractionated on a reverse phase HPLC column, and analyzed by electrospray ionization mass spectrometry. Altogether, 53 neuropeptides or other peptides derived from secretory pathway proteins were identified and sequenced using tandem mass spectrometry. These peptides arise from 21 distinct proteins: proenkephalin, proopiomelanocortin, prodynorphin, protachykinin A and B, procholecystokinin, promelanin‐concentrating hormone, proneurotensin, proneuropeptide Y, provasopressin, pronociceptin/orphanin, prothyrotropin‐releasing hormone, cocaine‐ and amphetamine‐regulated transcript, chromogranin A and B, secretogranin II, prohormone convertase 1 and 2, propeptidyl‐amidating monooxygenase, and proteins designated proSAAS and VGF. Approximately one third of the peptides found in wild‐type mice were not detectable in PC2 knock‐out mice, and another third were present at levels ranging from 25 to 75% of wild‐type levels. Comparison of the cleavage sites suggests that sequences with a Trp, Tyr and/or Pro in the P1′ or P2′ position, or a basic residue in the P3 position, are preferentially cleaved by PC2 and not by other enzymes present in the secretory pathway.

Neuropeptidomic analysis establishes a major role for prohormone convertase-2 in neuropeptide biosynthesis

J. Neurochem. (2010) 112, 1168–1179.

PAPA-1 Is a Nuclear Binding Partner of IGFBP-2 and Modulates Its Growth-Promoting Actions

IGF-binding proteins (IGFBPs) have multiple cellular effects, which occur by both IGF-dependent and -independent mechanisms. IGFBP-2 is involved in the regulation of both normal and carcinogenic cell growth. To further understand the actions of IGFBP-2, we carried out a yeast two-hybrid screen to search for intracellular partner proteins using a human prostate cDNA library. We isolated Pim-1-associated protein-1 (PAP-1)-associated protein-1 (PAPA-1) as an IGFBP-2-binding protein, whose expression and subcellular localization is regulated by both IGFBP-2 and androgens. Coimmunoprecipitation and glutathione S-transferase pull-down assay confirmed the interaction in vitro, and confocal microscopy showed the colocalization of IGFBP-2 and PAPA-1 in the nucleus. Suppression of PAPA-1 by small interfering RNA treatment enhanced the growth-promoting effect of IGFBP-2. Conversely, IGFBP-2-promoted bromodeoxyuridine incorporation into LNCaP cells was abrogated by the simultaneous overexpression of myc-hPAPA-1. Mouse embryonic fibroblasts from IGFBP-2 knockout mouse showed diminished growth activity compared with wild type, and expression of FLAG-mPAPA-1 decreased cell proliferation in IGFBP-2 knockout, but not control mouse embryonic fibroblasts. These studies suggest that the growth-promoting role of IGFBP-2 in prostate cancer is inhibited by its intracellular interaction with PAPA-1.

IGFBP-3 and TNF-α regulate retinal endothelial cell apoptosis.

PURPOSE We hypothesized that loss of insulin-like growth factor binding protein 3 (IGFBP-3) signaling would produce neuronal changes in the retina similar to early diabetes. METHODS To understand better the role of IGFBP-3 in the retina, IGFBP-3 knockout (KO) mice were evaluated for neuronal, vascular, and functional changes compared to wild-type littermates. We also cultured retinal endothelial cells (REC) in normoglycemia or hyperglycemia to determine the interaction between IGFBP-3 and TNF-α, as data indicate that both proteins are regulated by β-adrenergic receptors and respond antagonistically. We also treated some cells with Compound 49b, a novel β-adrenergic receptor agonist we have reported previously to regulate IGFBP-3 and TNF-α. RESULTS Electroretinogram analyses showed decreased B-wave and oscillatory potential amplitudes in the IGFBP-3 KO mice, corresponding to increased apoptosis. Retinal thickness and cell numbers in the ganglion cell layer were reduced in the IGFBP-3 KO mice. As expected, loss of IGFBP-3 was associated with increased TNF-α levels. When TNF-α and IGFBP-3 were applied to REC, they worked antagonistically, with IGFBP-3 inhibiting apoptosis and TNF-α promoting apoptosis. Due to their antagonistic nature, results suggest that apoptosis of REC may depend upon which protein (IGFBP-3 versus TNF-α) is active. CONCLUSIONS Taken together, loss of IGFBP-3 signaling results in a phenotype similar to neuronal changes observed in diabetic retinopathy in the early phases, including increased TNF-α levels.

Embryonic gene expression and pro‐protein processing of proSAAS during rodent development

In vitro assays have demonstrated that peptides derived from the recently–identified proSAAS precursor inhibit prohormone convertase 1 (PC1) suggesting that this novel peptide may function as an endogenous inhibitor of PC1. To further understand the role of proSAAS in vivo, we have investigated the expression of proSAAS mRNA and processing of proSAAS during pre‐ and early postnatal rodent development. In situ hybridization showed that, by embryonic day 12.5 (e12.5) in the rat, proSAAS mRNA was present in essentially all differentiating neurons in the mantle layer of the myelencephalon, metencephalon, diencephalon, spinal cord and several sympathetic ganglia. During later stages of prenatal development, widespread proSAAS expression continues in post‐mitotic neurons of both the CNS and PNS and begins in endocrine cells of the anterior and intermediate pituitary. Although proSAAS expression overlaps with PC1 in several regions, its overall expression pattern is significantly more extensive, suggesting that proSAAS may be multifunctional during development. Processed forms of proSAAS are present by at least mid‐gestation with marked accumulation of two C‐terminal forms, comprising the PC1 inhibitory fragment of proSAAS.

Targeted mutagenesis of processing enzymes and regulators: Implications for development and physiology

Neuroendocrine peptides are translated usually as inactive precursor propeptides that require posttranslational processing to be activated (Zhou et al., 1999). The identities of substrates known to require processing include important neurotrophic, growth, and developmental factors such as pro brain-derived neurotrophic factor (BDNF), proinsulin, insulin-like growth factor 1, transforming growth factor (TGF) and its receptor, and the family of bone morphogenic proteins (BMPs). Activation of propeptides requires limited endoproteolysis of the precursor substrate by prohormone convertases (PCs) (Zhou et al., 1999), followed by removal of C-terminal basic residues by members of the carboxypeptidase (CP) family (Fricker and Leiter, 1999). Subsequently, additional posttranslational modifications such as amidation, acetylation, sulfation, and phosphorylation may be utilized to impart full activation and function to the peptide substrate. Gene targeting in the mouse is an extremely powerful approach to investigate specific functions of genes in vivo. As discussed below, deletion of not only peptide precursors (e.g., adrenomedullin) (Caron and Smithies, 2001; Shindo et al., 2001; Shimosawa et al., 2002) but also the enzymes (e.g., furin) (Roebroek et al., 1998) responsible for processing these substrates can produce severe developmental abnormalities. Thus, both endocrine peptides and the specific enzymes responsible for their maturation have crucial, non-redundant roles in normal development. Consequently, proper spatial and temporal activation of processing enzymes (Zheng et al., 1994, 1997; Constam et al., 1996; Zheng and Pintar, 1997; Beck et al., 2002) becomes an important component in the regulation of embryogenesis. A multitude of neuroendocrine peptides in the central nervous system (CNS), peripheral nervous system (PNS), and pituitary are processed, including propituitary adenylate cyclase-activating polypeptide (PACAP), giving rise to amidated PACAP27 and PACAP38 (Kimura et al., 1990), procholecystokinin (proCCK), giving rise to several amidated CCK peptides (Mogensen et al., 1990), and proopiomelanocortin (POMC), which gives rise to several biologically active peptides including adrenocorticotropin (ACTH) and the opioid peptide -endorphin ( -EP) (Eipper and Mains, 1980). This review will briefly describe the roles of processing enzymes and related molecules as thus far deduced from studies of their developmental expression patterns and the mutant phenotypes of gene-targeted mice.

Strain-specific steroidal control of pituitary function

We have previously shown that 7B2 null mice on the 129/SvEvTac (129) genetic background die at 5 weeks of age with hypercorticosteronemia due to a Cushings-like disease unless they are rescued by adrenalectomy; however, 7B2 nulls on the C57BL/6NTac (B6) background remain healthy, with normal steroid levels. Since background exerts such a profound influence on the phenotype of this mutation, we have evaluated whether these two different mouse strains respond differently to high circulating steroids by chronically treating wild-type 129 and B6 mice with the synthetic steroid dexamethasone (Dex). Dex treatment decreased the dopamine content of the neurointermediate lobes (NIL) of 129 mice, leading to NIL enlargement and increased total D(2)R mRNA in the 129, but not the B6, NIL. Despite the decrease in this inhibitory transmitter, Dex-treated 129 mice exhibited reduced circulating alpha-melanocyte-stimulating hormone (alpha-MSH) along with reduced POMC-derived peptides compared with controls, possibly due to reduced POMC content in the NIL. In contrast, Dex-treated B6 mice showed lowered cellular ACTH, unchanged alpha-MSH and beta-endorphin, and increased circulating alpha-MSH, most likely due to increased cleavage of NIL ACTH by increased PC2. Dex-treated 129 mice exhibited hyperinsulinemia and lowered blood glucose, whereas Dex-treated B6 mice showed slightly increased glucose levels despite their considerably increased insulin levels. Taken together, our results suggest that the endocrinological response of 129 mice to chronic Dex treatment is very different from that of B6 mice. These strain-dependent differences in steroid sensitivity must be taken into account when comparing different lines of transgenic or knockout mice.