Daniel S. Straus

Nutritional regulation of hormones and growth factors that control mammalian growth.

Juvenile animals stop growing if they are fed a diet containing an inadequate amount of energy or protein. The molecular basis for this growth arrest is not completely understood. The cessation of growth that occurs in nutritionally restricted animals is not generally explained by a decrease in circulating growth hormone (GH). In most species, plasma GH is increased rather than decreased under conditions of nutritional restriction. Current evidence suggests that the biosynthesis of insulin‐like growth factor‐I (IGF‐I) is a key control point for nutritional regulation of growth. Plasma IGF‐I peptide levels and hepatic IGF‐I mRNA abundance are correlated with growth velocity and are consistently decreased when growth is arrested by nutritional deprivation. The decreased IGF‐I mRNA abundance observed in the fasting rat appears to be caused primarily by a decrease in IGF‐I gene transcription. In tissues and plasma, the insulin‐like growth factors are complexed with high‐affinity binding proteins, which are thought to modulate the tissue access and action of the IGFs. The hepatic mRNA abundance of two of the binding proteins (IGFBP‐1 and ‐2) is increased in nutritionally restricted animals. This increase in mRNA abundance is caused primarily by an increase in transcription of the IGFBP‐1 and IGFBP‐2 genes. Current research is focused on molecular mechanisms for regulation of IGF‐I and IGF‐binding protein gene expression.— Straus, D. S. Nutritional regulation of hormones and growth factors that control mammalian growth. FASEB J. 8: 6‐12; 1994.

Effect of amino acid limitation on the expression of 19 genes in rat hepatoma cells.

We showed previously that the abundance of serum albumin mRNA is decreased in H4‐II‐E rat hepatoma cells limited for a single essential amino acid (phenylalanine, methionine, leucine, or tryptophan). To define the specificity of this phenomenon, we examined the effect of amino acid limitation on the abundance of mRNAs for 19 genes in the H4‐II‐E cells. These genes included six genes whose expression is either completely liver‐specific or highly enriched in the liver compared with other tissues [albumin, transthyretin (TTR), transferrin, carbamyl phosphate synthetase‐I, urate oxidase, class I alcohol dehydrogenase], as well as a number of ubiquitously expressed “housekeeping” genes. The results indicated that the 19 genes could be divided into three classes based on their response to amino acid limitation. Class I genes (the six liver‐specific genes and α‐tubulin) exhibit decreased expression in response to amino acid limitation. The expression of class II genes [β2‐microglobulin, hypoxanthine‐guanine phosphoribosyl transferase (HPRT), H‐ferritin, ubiquitin (UbB), insulin‐like growth factor binding protein‐4, HNF‐1α] is not significantly affected by amino acid limitation. Class III genes [gadd153, β‐actin, ubiquitin (UbC), phosphoglycerate kinase‐1, C/EBPα, C/EBPβ] exhibit increased expression in response to amino acid limitation. Thus, specific inductive as well as repressive effects on gene expression are quite common in amino acid‐limited cells. The observation that all six genes whose expression is liver‐specific exhibited decreased expression in amino acid‐limited cells suggests a common mode of regulation of these genes by amino acid availability. The strong induction by amino acid limitation of the C/EBP inhibitor gadd153 is of interest in this regard, as increased levels of gadd153 could interfere with C/EBP, which is required for high expression of most liver‐specific genes. To investigate further the molecular mechanism for the decrease in albumin mRNA abundance, albumin nuclear transcript levels were quantified in control and tryptophan‐limited cells. Tryptophan limitation caused a decrease in albumin nuclear transcript abundance, and this decrease preceded the decrease in albumin mRNA, suggesting that the decrease in albumin mRNA was caused at least partly by a decrease in albumin gene transcription. Additional experiments with actinomycin D indicated that albumin mRNA was also destabilized in the tryptophan‐limited cells. Thus, the overall results indicate that the decrease in albumin mRNA in the tryptophan‐limited cells is caused by a specific decrease in albumin nuclear transcript abundance and destabilization of albumin mRNA.—Marten, N. W., Burke, E. J., Hayden, J. M., Straus, D. S. Effect of amino acid limitation on the expression of 19 genes in rat hepatoma cells. FASEB J. 8: 538‐544; 1994.

The peroxisome proliferator-activated receptor γ ligand rosiglitazone delays the onset of inflammatory bowel disease in mice with interleukin 10 deficiency

Aims: To test whether the peroxisome proliferator‐activated receptor &ggr; (PPAR&ggr;) ligand rosiglitazone (Ro) has therapeutic activity in the IL‐10−/− mouse model of inflammatory bowel disease (IBD), and to identify the cellular targets and molecular mechanisms of Ro action. Methods: The progression of spontaneous chronic colitis in IL‐10−/− mice was compared in 5‐week‐old mice fed a standard diet with or without Ro for 12 weeks. The possible therapeutic effect of Ro was also tested over a 6‐week interval in older IL‐10−/− mice with established IBD. Results: Treatment with Ro slowed the onset of spontaneous IBD in IL‐10−/− mice. Crypt hyperplasia, caused by increased mitotic activity of crypt epithelial cells, was also delayed by Ro. Treatment with Ro significantly decreased expression of interferon &ggr; (IFN&ggr;), interleukin 17 (IL‐17), tumor necrosis factor &agr;, and the inducible nitric oxide synthase mRNA in the colon, whereas expression of IL‐12p40 was unchanged. PPAR&ggr; was detected in epithelial cells throughout the crypts and surface. Ro increased expression of PPAR&ggr; protein in these cells, suggesting the existence of a positive feedback loop that would potentiate its action in these cells. Ro also specifically increased expression of a novel PPAR target, aquaporin‐8 (AQP8), in differentiated colonic epithelial surface cells, demonstrating that PPAR&ggr; is not only present but also regulates gene expression in these cells in vivo. Finally, Ro was ineffective in improving disease activity in older IL‐10−/− mice with established IBD. Conclusions: PPAR&ggr; is expressed, and the PPAR&ggr; ligand Ro regulates gene expression in colonic epithelial cells. As a single agent, Ro works best for disease prevention in the IL‐10−/− mouse model for IBD.

Differential Regulation of Chemokines by IL-17 in Colonic Epithelial Cells

The IL-23/IL-17 pathway plays an important role in chronic inflammatory diseases, including inflammatory bowel disease. In inflammatory bowel disease, intestinal epithelial cells are an important source of chemokines that recruit inflammatory cells. We examined the effect of IL-17 on chemokine expression of HT-29 colonic epithelial cells. IL-17 strongly repressed TNF-α-stimulated expression of CXCL10, CXCL11, and CCL5, but synergized with TNF-α for induction of CXCL8, CXCL1, and CCL20 mRNAs. For CXCL10, IL-17 strongly inhibited promoter activity but had no effect on mRNA stability. In contrast, for CXCL8, IL-17 slightly decreased promoter activity but stabilized its normally unstable mRNA, leading to a net increase in steady-state mRNA abundance. IL-17 synergized with TNF-α in transactivating the epidermal growth factor receptor (EGFR) and in activating ERK and p38 MAPK. The p38 and ERK pathway inhibitors SB203580 and U0126 reversed the repressive effect of IL-17 on CXCL10 mRNA abundance and promoter activity and also reversed the inductive effect of IL-17 on CXCL8 mRNA, indicating that MAPK signaling mediates both the transcriptional repression of CXCL10 and the stabilization of CXCL8 mRNA by IL-17. The EGFR kinase inhibitor AG1478 partially reversed the effects of IL-17 on CXCL8 and CXCL10 mRNA, demonstrating a role for EGFR in downstream IL-17 signaling. The overall results indicate a positive effect of IL-17 on chemokines that recruit neutrophils (CXCL8 and CXCL1), and Th17 cells (CCL20). In contrast, IL-17 represses expression of CXCL10, CXCL11, and CCR5, three chemokines that selectively recruit Th1 but not other effector T cells.

Effects of insulin on cellular growth and proliferation

Abstract Insulin stimulates the growth and proliferation of a variety of cells in culture. The growth-stimulatory effects of insulin are observed in Go/Gl arrested cells limited for serum growth factors or essential nutrients, and in cells growing in hormone-supplemented serum-free media. Some, but not all, of the effects of insulin on growth require superphysiological concentrations of insulin. The action of insulin on growth is synergistic with the action of other hormones and growth factors, including FGF, PDGF, PGF2α and vasopressin. This observation, as well as other observations regarding the temporal sequence of action of growth factors, suggests that different growth factors act on different intracellular biochemical events. Several hypotheses have been proposed to explain the effect of insulin on cellular proliferation, including regulation of essential metabolic processes and interaction of insulin with receptors for insulin-like growth factors. Evidence supporting these various hypotheses is reviewed. In addition to the growth-stimulatory effect of insulin observed in cell culture, a number of clinical examples suggest that insulin is an important growth-regulating hormone during fetal development.

Peroxisome proliferator–activated receptor δ limits the expansion of pathogenic Th cells during central nervous system autoimmunity

Peroxisome proliferator–activated receptors (PPARs; PPAR-α, PPAR-δ, and PPAR-γ) comprise a family of nuclear receptors that sense fatty acid levels and translate this information into altered gene transcription. Previously, it was reported that treatment of mice with a synthetic ligand activator of PPAR-δ, GW0742, ameliorates experimental autoimmune encephalomyelitis (EAE), indicating a possible role for this nuclear receptor in the control of central nervous system (CNS) autoimmune inflammation. We show that mice deficient in PPAR-δ (PPAR-δ−/−) develop a severe inflammatory response during EAE characterized by a striking accumulation of IFN-γ+IL-17A− and IFN-γ+IL-17A+ CD4+ cells in the spinal cord. The preferential expansion of these T helper subsets in the CNS of PPAR-δ−/− mice occurred as a result of a constellation of immune system aberrations that included higher CD4+ cell proliferation, cytokine production, and T-bet expression and enhanced expression of IL-12 family cytokines by myeloid cells. We also show that the effect of PPAR-δ in inhibiting the production of IFN-γ and IL-12 family cytokines is ligand dependent and is observed in both mouse and human immune cells. Collectively, these findings suggest that PPAR-δ serves as an important molecular brake for the control of autoimmune inflammation.

Effects of cyclopentenone prostaglandins and related compounds on insulin-like growth factor-I and Waf1 gene expression.

The molecular pathways by which the cyclopentenone prostaglandins (PGA and PGJ series) inhibit cell growth and tumorigenicity are poorly understood. These cellular responses may be caused by specific regulation of growth-related and stress-induced genes. A variety of prostaglandins were tested for their ability to regulate insulin-like growth factor-I (IGF-I) and Waf1 gene expression in C6 rat glioma cells. The prostaglandins (in order of potency) PGJ2 > PGA1 > PGA2, approximately PGD2 >> PGE2 all significantly repressed IGF-I gene expression. With the exception of PGE2, the same prostaglandins that repressed IGF-I also induced Waf1 gene expression. However, the order of potency for Waf1 induction was different than for IGF-I repression: PGA2 > PGA1 approximately PGJ2 > PGD2. The different order of potency of the prostaglandins in regulating IGF-I and Waf1 gene expression suggests that different intracellular signals may be involved in regulating the two genes. Augmentation of glutathione levels by pretreatment of cells with N-acetyl-L-cysteine attenuated the effect of PGA2 on IGF-I and Waf1 gene expression. conversely, depletion of the intracellular glutathione pool by pretreatment with buthionine sulfoximine potentiated the effect of PGA2 on the expression of both genes. These results suggest that conjugation with glutathione prevents the regulation of gene expression by PGA2. We also tested the effect of several simpler compounds that contain a five-membered ring system on IGF-I and Waf1 gene expression. 2-Cyclopenten-1-one, but not cyclopentene or cyclopentene, repressed IGF-I and induced Waf1 gene expression, demonstrating the requirement for an alpha, beta-unsaturated carbonyl for regulation of the two genes. The dione compound 4-cyclopentene-1,3-dione, which has two potentially reactive carbons rather than one, was considerably more potent than 2-cyclopentene-1-one in repressing IGF-I gene expression (IC50 = 30 microM for 4-cyclopentene-1,3-dione as compared with 167 microM for 2-cyclopentene-1-one). Additional results indicated that diethyl maleate, which has two alpha,beta-unsaturated carbonyls in a non-cyclic configuration, also repressed IGF-I gene expression (IC50 = 214 microM) and induced Waf1 gene expression, indicating that the cyclic structure is not required for either effect.

Bacteria and Bacterial rRNA Genes Associated with the Development of Colitis in IL-10-/-Mice

Background: Microorganisms appear to play important yet ill‐defined roles in the etiology of inflammatory bowel disease (IBD). This study utilized a novel population‐based approach to identify bacteria and bacterial rRNA genes associated with the development of colitis in IL‐10−/− mice. Methods: Mice were housed in 2 environments: a community mouse facility where the mice were fed nonsterile chow (Room 3) and a limited access facility where the mice were fed sterile chow (Room 4). Every month the disease activity levels were assessed and fecal bacterial compositions were analyzed. At the end of the experiments histological and bacterial analyses were performed on intestinal tissue. Results: Although disease activity increased over time in both environments, it progressed at a faster rate in Room 3 than Room 4. Culture and culture‐independent bacterial analyses identified several isolates and phylotypes associated with colitis. Two phylotypes (GpC2 and Gp66) were distinguished by their negative associations with disease activity in fecal and tissue samples. Notably, rRNA genes from these phylotypes had high sequence identity (99%) to an rRNA gene from a previously described flagellated Clostridium (Lachnospiraceae bacterium A4). Conclusions: The negative associations of these 2 phylotypes (GpC2 and Gp66) suggest that these bacteria were being immunologically targeted, consistent with prior findings that the Lachnospiraceae bacterium A4 bears a prevalent flagellar antigen for disease‐associated immunity in murine immune colitis and human Crohns disease. Identification of these associations suggests that the experimental approach used in this study will have considerable utility in elucidating the host–microbe interactions underlying IBD.

Effects of bradykinin on DNA synthesis in resting NIL8 hamster cells and human fibroblasts

Bradykinin stimulates [3H]thymidine incorporation and DNA synthesis in resting, serum-deprived NIL8 hamster cells. The ED50 for this stimulation is 4.52 +/- 2.91 nM. Other kinin peptides including lys-bradykinin (kallidin) and met-lys-bradykinin also stimulate [3H]thymidine incorporation in the NIL8 cells, whereas desarg9-bradykinin is without effect, suggesting action of the kinin peptides through type B2 receptors. Bradykinin also stimulates DNA synthesis in IMR-90 human fibroblasts; however, this effect is observed only in the presence of indomethacin, which blocks prostaglandin synthesis. These results suggest that prostaglandins act as negative modulators of the growth-stimulatory effects of bradykinin in the fibroblasts. This conclusion is supported by the observation that exogenously added PGE1, PGE2, PGA1, PGA2, PGB1, and PGB2 strongly inhibit [3H]thymidine incorporation in the human fibroblasts. The direct effect of bradykinin observed in the NIL8 cells may be attributable to the relative resistance of these cells to growth inhibition by prostaglandins.

Apical and basolateral pools of proteinase-activated receptor-2 direct distinct signaling events in the intestinal epithelium.

Studies suggest that there are two distinct pools of proteinase-activated receptor-2 (PAR₂) present in intestinal epithelial cells: an apical pool accessible from the lumen, and a basolateral pool accessible from the interstitial space and blood. Although introduction of PAR₂ agonists such as 2-furoyl-LIGRL-O-NH₂ (2fAP) to the intestinal lumen can activate PAR₂, the presence of accessible apical PAR₂ has not been definitively shown. Furthermore, some studies have suggested that basolateral PAR₂ responses in the intestinal epithelium are mediated indirectly by neuropeptides released from enteric nerve fibers, rather than by intestinal PAR₂ itself. Here we identified accessible pools of both apical and basolateral PAR₂ in cultured Caco2-BBe monolayers and in mouse ileum. Activation of basolateral PAR₂ transiently increased short-circuit current by activating electrogenic Cl⁻ secretion, promoted dephosphorylation of the actin filament-severing protein, cofilin, and activated the transcription factor, AP-1, whereas apical PAR₂ did not. In contrast, both pools of PAR₂ activated extracellular signal-regulated kinase 1/2 (ERK1/2) via temporally and mechanistically distinct pathways. Apical PAR₂ promoted a rapid, biphasic PLCβ/Ca²(+)/PKC-dependent ERK1/2 activation, resulting in nuclear localization, whereas basolateral PAR₂ promoted delayed ERK1/2 activation which was predominantly restricted to the cytosol, involving both PLCβ/Ca²(+) and β-arrestin-dependent pathways. These results suggest that the outcome of PAR₂ activation is dependent on the specific receptor pool that is activated, allowing for fine-tuning of the physiological responses to different agonists.