Somatostatin

Somatostatin David E. Elliott* Division of Gastroenterology, Department of Medicine, University of Iowa, 4611 JCP, Iowa City, IA 52242, USA * corresponding author tel: 319-353-8574, fax: 319-353-6399, e-mail: [email protected] DOI: 10.1006/rwcy.2001.13008.

SUMMARY

Figure 1 Preprosomatostatin.

Somatostatin is a small cyclic peptide with antiinflammatory and immunoregulatory properties. It is made by nerve, endocrine, and inflammatory cells. Somatostatin and somatostatin agonists inhibit inflammation in several animal model systems and in human diseases. Somatostatin acts on T cells to inhibit antigen-stimulated IFN release. It is made by activated macrophages within inflammatory lesions. Cytokines such as IFN or IL-10 and inflammatory mediators such as LPS or prostaglandin E2 induce normal splenic macrophages to make somatostatin. Somatostatin is a highly conserved peptide that functions as a neurotransmitter, a hormone, and an inducible immunoregulatory cytokine.

BACKGROUND

Discovery Somatostatin was discovered in 1973 (Brazeau et al., 1973) as a somatotropin (growth hormone) release inhibiting factor (SRIF). Somatostatin is abbreviated SST, SOM, or SRIF. Somatostatin-producing cells have been identified throughout the body, where it functions as a neurotransmitter, a hormone, or a cytokine-like paracrine/autocrine regulator of cell function (Patel, 1999). Somatostatin is made by all vertebrate, and some invertebrate and plant species. Somatostatin is initially synthesized as an inactive preprosomatostatin consisting of 116 amino acids (Figure 1). The active peptide is located at the Cterminus of the prepromolecule. The 24 amino acid leader sequence is cleaved in the endoplasmic reticulum to yield a 92 amino acid prosomatostatin protein. Two

Cytokine Reference

different forms of mature somatostatin exist. Prosomatostatin can be cleaved into either a 28 amino acid form (SOM 28) or a 14 amino acid form (SOM 14). It is the 14 amino acid peptide that is immunoregulatory.

Alternative names SOM, SOM-14, SST, SST-14, SRIF.

Structure Somatostatin is a cyclic peptide due to a cystine thiol bond (Figure 2). The thiol bond helps to hold phenylalanine, tryptophan, lysine, and threonine in conformation. This amino acid loop is present in octreotide (SMS 201±995, Sandostatin), which is a stable agonist of somatostatin. The formula for octreotide is: H-(D)Phe-Cys-Phe-(D)Trp-Lys-Thr-Cys-Thr-ol, (the cysteines are crosslinked). Somatostatin agonists have clinical utility, prompting analysis of the pharmacology

Copyright # 2001 Academic Press

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David E. Elliott Figure 2 Somatostatin 14.

and structure of somatostatin and its agonists. There are five different receptors for somatostatin. The precise stereospecific requirements of ligands for each receptor is being elucidated.

Main activities and pathophysiological roles Somatostatin is highly conserved in vertebrates and regulates secretion of many hormones such as growth hormone, calcitonin, insulin, gastrin, and renin. Clinically, somatostatin agonists are used to control aberrant hormone production. Somatostatin also regulates inflammation and T cell function. Importantly, somatostatin is made at sites of inflammation (Weinstock et al., 1990). Inflammatory mediators and cytokines can induce somatostatin production by murine macrophages. Somatostatin and agonists of somatostatin inhibit inflammatory reactions in mice (Blum et al., 1992; Bowman et al., 1996), rats (Karakalis et al., 1994, rabbits (MatucciCerinic et al., 1995), and humans (Coari et al., 1995; Fioravanti et al., 1995; Matucci-Cerinic et al., 1988; Krassas et al., 1997). Thus, somatostatin functions in a feedback mechanism to regulate inflammatory reactions. T cells constitutively express receptors for somatostatin (see SSTR2 chapter). Somatostatin inhibits murine T cell release of IFN (Blum et al., 1992; Elliott et al., 1999). Somatostatin regulates IFN mediated TH1 effects such as B cell switching to IgG2a in mice (Blum et al., 1993). Thus, somatostatin regulates TH1/TH2 circuitry.

GENE AND GENE REGULATION

Accession numbers Human somatostatin gene: J00306 (Shen and Rutter, 1984)

Human preprosomatostatin mRNA: J00306 (Shen et al., 1982) Mouse preprosomatostatin gene: X51468 (Fuhrmann et al., 1990) Rat: J00787 Pig: P01168 Sheep: AF031488 Cow: M31217 Cat: L42325 Chicken: X60191 Frog: U68136 Lungfish: AF126243 Goldfish: U40754 Catfish: M25903

Sequence For human gene and mRNA sequences see Figure 3 from accession number J00306. Features of the human somatostatin gene on chromosome 3q28 are: 50 flank (promoter region): 1±1125 Transcriptional start site: 1126 CDS: (1231±1368) joined to (2246±2458) Exon 1: 1231±1368 Intron 1: 1369±2245 Exon 2: 2246±2605 Somatostatin 28 sequence: 2372±2455 Somatostatin 14 sequence: 2414±2455 For mouse preprosomatostatin gene and mRNA sequences see Figure 4. Features of the mouse gene are: 50 flank (promoter region): 1±795 Transcriptional start site: 796 CDS: (896±1033) joined to (1699±1911) Exon 1: 896±1033 Intron 1: 1034±1698 Exon 2: 1699±2059

Chromosome location Human gene is on chromosome 3q28.

PROTEIN

Accession numbers Human somatostatin: J00306 Mouse preprosomatostatin: X51468 Rat: J00787 Pig: P01168 Sheep: AF031488 Cow: M31217 Cat: L42325

Somatostatin 3 Figure 3 Nucleotide sequences for the human somatostatin gene on chromosome 3q28. Lower case is not transcribed. Uppercase is transcribed. Protein-coding sequence shown in bold.

Chicken: X60191 Frog: U68136 Lungfish: AF126243 Goldfish: U40754 Catfish: M25903

Sequence See Figure 5.

Figure 4 Nucleotide sequences for the mouse preprosomatostatin gene (from X51468) and mRNA. Lower case is not transcribed. Uppercase is transcribed. Protein-coding sequence shown in bold. TATA box shown underlined.

Figure 5 Amino acid sequence for proprosomatostatin protein deduced from mRNA sequence.

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David E. Elliott

Important homologies Somatostatin is highly conserved, sharing a great deal of homology between species. Two genes for somatostatin exist in fish, probably due to gene duplication. Table 1 includes the fish gene with most homology to the mammalian gene.

CELLULAR SOURCES AND TISSUE EXPRESSION

Cellular sources that produce Somatostatin is made in many tissues. The brain, hypothalamus, thymus, and intestine are particularly rich sources. Splenocytes from normal mice normally do not make somatostatin. However, macrophages within the spleen are induced to make somatostatin by some cytokines and inflammatory mediators (Blum et al., 1998). Somatostatin is made in spleens from animals with chronic inflammation (e.g. schistosomiasis).

Eliciting and inhibitory stimuli, including exogenous and endogenous modulators Transcription of the somatostatin gene is regulated. The gene sequence shown in Figure 3 and Figure 4

includes 795 bp of the promoter region proximal to the transcription start site. Immediately before the noncanonical TATA box is the GAGA box (AGAGAGAGA) that controls somatostatin expression in endocrine cells. Adjacent to the GAGA box is a CREB (cyclic AMP responsive element binding) site (TGACGTCA) that confers cAMP-initiated transcription. We showed (Blum et al., 1998; Elliott et al., 1998) that macrophages from normal mice make and release somatostatin after 4 hours exposure to IFN (200 U/mL), IL-10 (30 ng/mL), LPS (30 g/mL), PGE2 (110ÿ6 M), or a cAMP analog (110ÿ4 M). Thus, macrophages make and release somatostatin in response to several cytokines and inflammatory mediators. Prostaglandin and cAMP analogs are probably inducing transcription via CREB interactions. LPS likely signals through NFB. The signaling pathways utilized by IFN and IL-10 to induce macrophage somatostatin transcription is currently being studied. Importantly, the somatostatin mRNA level is also negatively regulated. The neuropeptide/cytokine substance P decreases somatostatin transcripts in splenic macrophages stimulated with IL-10 or IFN (Blum et al., 1998). In contrast to somatostatin, substance P promotes IFN release by antigen-stimulated T cells. Substance P prevents production of somatostatin that would serve to inhibit T cell IFN release. IL-4 alone does not induce somatostatin transcription but it prevents substance P-mediated decrease in somatostatin mRNA. Thus, TH1/TH2 cytokines regulate macrophage somatostatin production.

Table 1 Percentage identity of somatostatin protein between species Animal

Accession number

Preprosomatostatin

Prosomatostatin

Somatostatin 14

Mouse (Mus musculus)

X51468

96

98

100

Rat (Rattus norvegicus)

J00787

96

98

100

Pig (Sus scrofa)

P01168

±

100

100

Sheep (Ovis aries)

AF031488

99

100

100

Cow (Bos taurus)

M31217

98

99

100

Cat (Canis familiaris)

L42325

98

100

100

Chicken (Gallus gallus)

X60191

87

91

100

Frog (Rana ridibunda)

U68136

76

84

100

Lungfish (P. annectens)

AF126243

72

78

100

Goldfish (C. auratus)

U40754

68

73

100

Catfish (I. punctatus)

M25903

58

61

100

Somatostatin 5

RECEPTOR UTILIZATION There are five different somatostatin receptors named SSTR1 to SSTR5. Human and murine inflammatory cells express SSTR2.

IN VITRO ACTIVITIES

In vitro findings Somatostatin and octreotide inhibit antigen-induced IFN release from granuloma and splenic T cells of mice with schistosomiasis (Blum et al., 1992; Elliott et al., 1999). This effect is most substantial when T cells are stimulated with low doses of antigen. The physiologic effects of IFN inhibition by somatostatin and octreotide is reflected in the reduction of splenic and granuloma B cell IgG2a production in vitro and in vivo (Blum et al., 1993; Elliott et al., 1999). Treatment with exogenous somatostatin or octreotide regulates granulomatous inflammation in murine schistosomiasis. Importantly, somatostatin is also made in the granulomas. Granuloma macrophages synthesize and release somatostatin 14 (Weinstock et al., 1990; Elliott et al., 1998). Normal splenic macrophages do not make somatostatin constitutively but will transcribe and synthesize the peptide after 4 hours exposure to IFN , IL-10, LPS, PGE2, or cAMP analog. Thus, somatostatin functions as an inducible cytokine to downregulate T cell function. Somatostatin may regulate many different cell types within the granuloma cell population. For example, somatostatin may influence antigen-presenting cells to inhibit T cell IFN release. However, somatostatin does act directly on T cells. Somatostatin inhibits IFN release by cloned D1.1 T cells stimulated with crosslinked monoclonal anti-CD3 and anti-CD4 in the absence of antigen-presenting cells (Elliott et al., 1999).

Bioassays used Tissue and cellular somatostatin production is measured by acid extraction, HPLC fractionation, and radioimmunosorbant assay (RIA) on the HPLC fractions to obtain a quantitative measurement (Weinstock et al., 1990; Blum et al., 1998). Somatostatin can be identified in cells by immunohistochemistry using monoclonal antibody CURE.S607 (Blum et al., 1998; Wong et al., 1990). Murine preprosomatostatin mRNA can be measured using a quantitative competitive RT-PCR assay (Elliott et al., 1998).

IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS

Normal physiological roles In addition to controlling growth hormone release, somatostatin regulates many diverse physiologic functions. The list of regulatory functions includes inhibition of: thyroid-stimulating hormone release, TSH-mediated thyroxine release, parafollicular cell calcitonin release, pancreatic islet hormone secretion (e.g. insulin), renin release by the juxtaglomerular cells of the kidney, stomach G-cell gastrin production (thereby regulating gastric acid production), and bile production. In general, somatostatin inhibits endocrine and exocrine activity of many cell types. Somatostatin decreases gall bladder contractility, gastric emptying, and segmental intestinal motility but stimulates the intestinal migrating motor complex. Somatostatin suppresses proliferation of many cell types including cancers. The ability of this small peptide to have diverse functions is explained by the presence of five different somatostatin receptors. T cells, B cells, and macrophages express somatostatin receptor subtype 2 (SSTR2). Somatostatin 14 is an immunoregulatory peptide. Somatostatin regulates the granulomatous response of schistosome-infected mice. Mice infected with schistosomes develop granulomas around the parasite eggs that lodge in the liver and intestine (schistosomiasis) (Elliott, 1996). Mice treated in vivo with octreotide (a stable SOM14 agonist) develop granulomas 60% the size that form in sham-treated mice (Blum et al., 1992). Somatostatin can regulate inflammation in several other model systems. Somatostatin suppressed the carrageenan inflammatory reaction in rats (Karalis et al., 1994). Treatment with somatostatin delayed onset of diabetes in the NOD-scid/scid transfer model of murine insulin-dependent diabetes mellitus (IDDM) (Bowman et al., 1996). Intra-articular injection of somatostatin reduced established fibrin-induced arthritis in rabbits (Matucci-Cerinic et al., 1995). Octreotide treatment decreased colonic mucosal platelet-activating factor, and leukotriene B4 content and inhibited mucosal damage in rats with acetic acid-induced acute colitis (Eliakim et al., 1993). Administration of octreotide decreased colonic TNF, IL-1 , and IFN in the rat trinitrobenzene sulfonic acid (TNBS) model of colitis (Lamrani et al., 1999). Somatostatin and its analogs reduce inflammation in patients with rheumatoid arthritis, psoriatic arthritis, and Graves' disease.

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IN THERAPY

Preclinical ± How does it affect disease models in animals? Agonists Somatostatin has been used in patients with inflammatory diseases. Intra-articular injection of somatostatin reduces synovitis in patients with rheumatoid arthritis (Coari et al., 1995; Fioravanti et al., 1995). Somatostatin infused over 48 hours reduced polyarticular joint pain and skin lesions in 12 of 18 patients with psoriatic arthritis (Matucci-Cerinic et al., 1988). Lanreotide, a long-acting somatostatin analog, significantly improved the inflammatory ocular manifestations of Graves' disease (Krassas et al., 1997). Stable analogs of somatostatin are used clinically to manage several other diseases and conditions. These include acromegaly (Newman, 1999), carcinoid syndrome (Oberg, 1998), chronic diarrhea (Farthing, 1996), and bleeding esophageal varices (Hadengue, 1999). Somatostatin suppresses growth of nonendocrine neoplasms in several model systems (Hofland and Lamberts, 1997; Pollak and Schally, 1998). Unfortunately, clinical trials have not shown efficacy for somatostatin analogs in treating breast cancer (O'Byrne et al., 1999; Ingle et al., 1999). Many endocrine and nonendocrine neoplasms avidly bind radiolabeled somatostatin analogs. This allows the scintigraphic localization of primary and metastatic lesions to aid surgical resection (Kwekkeboom and Krenning, 1997). Recently, intensely labeled analogs have been used to concentrate radioisotopes at the site of malignancies. Scintigraphy with radiolabeled somatostatin agonists demarcates areas of granulomatous inflammation in patients with diseases such as Wegener's granulomatosis, tuberculosis, sarcoidosis, and aspergillosis (Ozturk et al., 1994; van Hagen et al., 1994a, 1994b, 1994c; Postema et al., 1996). This demonstrates that cells within an inflammatory reaction express receptors for somatostatin (John et al., 1996). Antagonists Antagonists for somatostatin have been identified (Bass et al., 1996; Hocart et al., 1998) but have not been clinically applied.

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ACKNOWLEDGEMENTS Grants from the National Institutes of Health (DK02428).