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FASEB J. Jul 2009; 23(7): 2024–2033.
PMCID: PMC2704596

Structure, expression, and biological function of INSM1 transcription factor in neuroendocrine differentiation

Michael S. Lan*‡,1 and Mary B. Breslin*§

Abstract

Zinc-finger transcription factors are DNA-binding proteins that are implicated in many diverse biological functions. INSM1 (formerly IA-1) contains five zinc-finger motifs and functions as a transcription factor. INSM1 protein structure is highly conserved in homologues of different species. It is predominantly expressed in developing neuroendocrine tissues and the nervous system in mammals. INSM1 represents an important player in early embryonic neurogenesis. In pancreatic endocrine cell differentiation, Ngn3 first activates INSM1 and subsequently NeuroD/β2. Conversely, INSM1 exerts a feedback mechanism to suppress NeuroD/β2 and its own gene expression. INSM1 gene ablation in the mouse results in the impairment of pancreatic endocrine cell maturation. Further, deletion of INSM1 severely impairs catecholamine biosynthesis and secretion from the adrenal gland that results in early embryonic lethality. Genetically, INSM1 acts as a downstream factor of Mash 1 and Phox2b in the differentiation of the sympatho-adrenal lineage. In the developing neocortex, mouse embryos lacking INSM1 expression contain half the number of basal progenitors and show a reduction in cortical plate radial thickness. Cell signaling studies reveal that INSM1 contributes to the induction of cell cycle arrest/exit necessary to facilitate cellular differentiation. INSM1 is highly expressed in tumors of neuroendocrine origin. Hence, its promoter could serve as a tumor-specific promoter that drives a specific targeted cancer gene therapy for the treatment of neuroendocrine tumors. Taken together, all of these features of INSM1 strongly support its role as an important regulator during neuroendocrine differentiation.—Lan, M. S., Breslin, M. B. Structure, expression, and biological function of INSM1 transcription factor in neuroendocrine differentiation.

Keywords: Ngn3, Mash 1, zinc-finger transcription factor, pancreatic endocrine pathway, sympatho-adrenal lineage development, developing neocortex

In the early 1990s, a subtraction library was constructed using rare human insulinoma and glucagonoma tissues (National Institutes of Health; ref. 1). This unique strategy was designed to reveal specific antigens that may be associated with insulinoma and/or polyendocrinoma as well as encode autoantigens in type 1 diabetes. A series of clones was identified from this subtraction library and named as insulinoma-associated-1, -2, etc. (IA-1, -2, · · ·). The most well-known antigen identified in this subtraction library was IA-2, which represents a major autoantigen in type 1 diabetes (2). IA-1 was the first clone identified by this technique and revealed a restricted tissue expression pattern. Analysis of the IA-1 primary sequence revealed the presence of five zinc-finger DNA-binding motifs in the C terminus. Later, the IA-1 gene was renamed INSM1 (insulinoma associated-1) in the GenBank DNA database. INSM1 is mainly expressed in fetal neuroendocrine tissues and tumors of neuroendocrine origin. Therefore, extensive efforts have been focused on the elucidation of its biological function in neuroendocrine differentiation and transformation.

The neuroendocrine system involves the interaction between the nervous system and a variety of endocrine glands, which include the pituitary, thyroid, parathyroid, and adrenal glands, the ovaries and testes, the endocrine pancreas, the pineal gland, the gastrointestinal endocrine system, and the respiratory endocrine system. These endocrine cells play a pivotal role in maintaining the body’s metabolism by secreting potent hormones for target tissues in response to stress and injury, growth and development, absorption of nutrients, energy metabolism, water and electrolyte balance, reproduction, birth, and lactation. During development, these endocrine/neuroendocrine cells each follow a unique differentiation pathway. Abnormal differentiation and/or deregulation of these endocrine/neuroendocrine cells such as generation of neuroendocrine tumors have a profound effect on the body’s metabolism. Therefore, it is important to understand the molecular mechanisms required for the growth and differentiation of these hormone-producing cells. In this review, we introduce a neuroendocrine-specific zinc-finger transcription factor, INSM1, describe its structural features, its restricted expression pattern, and the recent identification of its biological functions in neuronal and neuroendocrine differentiation as it pertains to the regulation of the basal progenitors (BPs) of the mammalian neocortex, the endocrine pancreas, and the sympathoadrenal (SA) gland. These unique features imply that INSM1 is not only an important player in embryonic neuroendocrine differentiation but that also the tumor-specific promoter may be useful for the development of gene therapeutic interventions.

STRUCTURAL FEATURES OF INSM1

INSM1 was originally cloned from an insulinoma-glucagonoma subtraction library (1). It was mapped to chromosome 20p11.2 as an intronless gene (3). The cDNA clone has a 2838-bp sequence consisting of an open reading frame of 1530 nucleotides, which translates into a protein of 510 aa with a pI value of 9.1 and a predicted molecular mass of 52,923 Da (Fig. 1A). In the 3′-untranslated region, there are seven ATTTA sequences between two polyadenylation signals, which are postulated to be recognition signals for specific degradation of mRNA as reported for lymphokines, cytokines, and proto-oncogenes (4, 5). Based on the deduced protein sequence, INSM1 can be divided into two major domains. The amino-terminal domain (aa 1-250) contains a high percentage of proline, glycine, and alanine residues. Proline-rich (20–30%) sequences are found in many mammalian transcription factors and serve as protein-protein interacting domains that mediate both transcriptional activation and/or repression (6, 7). There are other additional features located in the amino-terminal sequence, a putative nuclear localization signal (NLS position 221-246), four dibasic amino acids (positions 8-9, 11-12, 221-222, and 227-228), and a potential amidation signal sequence, Pro-Gly-Lys-Arg (position 198–201). The dibasic amino acids are cleavage recognition sites for processing peptide hormone precursors, such as insulin, glucagon, somatostatin, and pancreatic polypeptide. An α-amide group is common to many bioactive neuroendocrine peptides. It is essential for the biological activity of pancreatic polypeptide as well as other amidated peptides (8, 9 ). Although INSM1 contains four dibasic amino acids and an amidation signal peptide suggesting that the amino-terminal portion of the INSM1 could be processed and amidated as a peptide hormone, no evidence thus far has shown that INSM1 protein is processed and amidated in neuroendocrine cells. The carboxyl-terminal sequence (aa 251-510) contains five putative Cys2-His2-type zinc-finger motifs. These five zinc-finger motifs are symmetrically spaced at the carboxy terminus. Two tandem repeated zinc-finger motifs from either end are spaced by 45/46 aa from the middle zinc finger (third). This exact spacing between the two tandem repeat zinc fingers suggests a precise mode of DNA binding. However, the first zinc-finger sequence, aa 266-293, is lacking the last His residue at position 289 and is replaced by Arg (Fig. 1B). The structural features of INSM1 strongly suggest that INSM1 is a zinc-finger DNA-binding protein. The functional role of INSM1 was further elucidated by the determination of the consensus DNA-binding sequence, TG/TC/TC/TT/AGGGGG/TCG/A, using a selected and amplified random oligonucleotide binding assay (10). Multiple potential target genes were subsequently revealed by searching the eukaryotic promoter database and included INSM1 itself (11). DNA-binding analysis of each zinc-finger motif showed that a construct containing zinc-finger 2 and 3 is sufficient to mimic the same activity as the intact 1-5 zinc-finger domain, indicating that zinc-fingers 2 and 3 contain all the information necessary to specifically modulate target gene binding (10).

Figure 1.
INSM1 structure. A) INSM1 mRNA is encoded by an intronless gene expanding a 2838-bp sequence with a 1530-bp coding sequence. The 510-aa protein can be divided into an N-terminal (aa 1-250) and a C-terminal (aa 251-510) domain. The N-terminal domain contains ...

EVOLUTIONARY HOMOLOGUES AMONG DIFFERENT SPECIES

Evolutionary studies based on comparison of the protein sequences of various species revealed that the INSM1 structure is 99.4% identical between humans and chimpanzees (Fig. 2). The degree of relatedness of mouse Insm1 to human is 90.6% identical at the protein level. The predicted rat Insm1 contains one less zinc-finger motif but shows a highly conserved protein sequence (76.7%). Comparison of human INSM1 with INSM1 homologues in Xenopus laevis, zebrafish, Drosophila melangaster, and Caenorhabditis elegans showed 55.7, 54.8, 22.6, and 18.2% identity, respectively. The majority of the homologues including human contains a potential nuclear localization signal upstream of zinc-finger 1 consistent with its role in the nucleus as a transcriptional regulator. The putative first zinc-finger motif contains a common Arg substitution for the last His residue in various species except D. melangaster, where Cys replaces the His residue. Both D. melangaster and C. elegans contain only three zinc-finger motifs in contrast to the other homologues. The most highly conserved motif is located in the second zinc finger, which scores a 96% identity among the different species. The high conservation of the second zinc-finger motif is particularly significant since human INSM1 zinc-fingers 2 and 3 were shown biochemically to be sufficient for target gene binding (10).

Figure 2.
Evolutionary conservation of INSM1 homologues. A) Schematic diagram of INSM1 homologues from human, chimpanzee, mouse, rat, X. laevis, zebrafish, D. melangaster, and C. elegans. Zinc-finger region represents a typical Cys2-His2-type zinc-finger motif. ...

C. elegans is a nematode used extensively as a model organism to study development. Approximately 35% of C. elegans genes have human homologues. Egg-laying defective-46 (Egl-46) is an INSM1 homologue in C. elegans. It encodes a protein of 286 aa with 3 equally spaced zinc-finger motifs. The first and third zinc fingers contain either Arg or Gln substitutions for the last His residue. The highly conserved second zinc finger predicts its transcription factor potential. A detailed expression study of egl-46 showed expression in ventral cord neurons, cells in the larvae head, Q neuroblasts [precursors to the anterior ventral microtubule (AVM) and posterior ventral microtubule (PVM) neurons], the touch receptor cells, and the touch (FLP) mechanosensory neurons. In all cases, expression was transient during the early larval stages (12). A study to determine genes involved in the function or development of the hermaphrodite-specific neuron (HSN), the egg-laying motor neuron, identified egl-44 and egl-46 (13, 14). Mutants in egl-46 had HSN, defects in cell migration, serotonin production, and axonal outgrowth (13). Due to a degree of overlap between genes expressed in specific cell types, a complex interaction between both positive and negative regulators in a cell determines its fate. The egl-46 is expressed in the FLP sensory neurons that sense touch to the nose required for avoidance response and touch receptor cells (15). Expression of egl-44 and egl-46 in FLP sensory neurons represses transcription of touch characteristics in these cells (12). Mutation in either the egl-44 or egl-46 gene results in a transformation of the FLP cells into cells that resemble the touch cells (12). C. elegans male hook (HOB) neurons are necessary for hermaphrodite vulva sensing during mating. The egl-46 is required for vulva location and HOB gene expression during differentiation (16). In C. elegans, egl-46 plays a critical role in the differentiation of a diverse group of neuronal cells.

D. melangaster is another model organism widely used to study developmental biology. A differential expression screen of a D. melangaster cDNA library prepared from 8.5-h-old (stage 11) embryonic heads identified nerfin-1, for nervous finger, as a neuronal precursor gene (17). Nerfin-1 is an INSM1 homologue in D. melangaster. During early central nervous system (CNS) development, nerfin-1 gene expression is activated in most of the neuroblasts before lineage formation. After early sublineage formation, nerfin-1 expression shifts to the ganglion mother cells but is not detected in mature neurons or glia (17). Despite the presence of nerfin-1 mRNA in many neuronal precursor cells, the nerfin-1 protein can be detected only transiently in a subset of neuronal precursor cells that will undergo a single final division to generate nascent neurons. A biochemical study (18) showed that the nerfin-1 mRNA is subject to post-transcriptional silencing by multiple microRNAs that regulate its spatial and temporal translation dynamics in the developing nervous system. This mechanism is potentially important for INSM1 in other species that could use the evolutionary conserved microRNA-binding site for the post-transcriptional regulation of gene expression. Functionally, nerfin-1 is a nuclear regulator of axon guidance and is required for a subset of early pathfinding events in the developing D. melangaster CNS. Although nerfin-1 null mutations displayed no detectable alterations in neuroblast lineage development, CNS axon patterning revealed defects in axon projections within the embryonic CNS but not in the peripheral nervous system. A screen of potential targets of nerfin-1 regulation identified a subset of genes involved in axon guidance (19).

Zebrafish (Danio rerio) is a vertebrate containing two INSM1-like genes, insm1a and insm1b, which are expressed in the developing nervous system (20). The timing of peak insm1a and insm1b expression corresponds precisely to the peak period of zebrafish neurogenesis. Their expression patterns resemble the other invertebrate homologues in early neurogenesis and in pancreatic progenitors, and their expression largely overlapped the expression pattern of deltaA, a proneural marker.

Medaka (Oryzias latipes) is a teleost that shares many common zebrafish characteristics. With the use of whole-mount in situ hybridization of the continuously growing optic tectum, genes were identified from a large number of cells that underwent proliferation, differentiation, and cell cycle exit. Ol-insm1a and Ol-insm1b showed strong expression in the growth arrest zone as well as the telencephalon, rhombic lips, midbrain tectum, hindbrain, pancreas, and differentiation zone of the retina. Studies to determine the function of these genes were performed by generating transgenic animals that overexpress Ol-insm1b in proliferating neural progenitors. Overexpression of Ol-insm1b caused a reduction in cell numbers resulting from cell cycle arrest in the absence of increased apoptosis. The zinc-finger domain of Ol-insm1b was required for this effect. Therefore, these studies (21) demonstrate a clear role of INSM1 in cell cycle exit for differentiation.

A recent study (22) demonstrated that the evolutionarily conserved INSM1 gene is expressed during X. laevis embryogenesis in neural plate primary neurons, as well as in uncharacterized anteroventral cells identified as noradrenergic neurons. Other regions with INSM1 expression were the neural tube along the entire A/P axis, forebrain, midbrain, pineal gland, cranial ganglia, eyes, and olfactory placodes. The X. laevis study focused on an uncharacterized population of cells located in the anteroventral regions of embryos, which they define as noradrenergic neurons. They showed that the formation of the anteroventral cells is dependent on bone morphogenic proteins (BMPs) and inhibited by Notch, a key component of the lateral inhibition signaling pathway. INSM1 expression in X. laevis noradrenergic primary neurons is regulated by Xash1, Phox2a, and Hand2, other essential components of the noradrenergic transcriptional network. INSM1 morpholinos demonstrate that it is required for noradrenergic neuron differentiation but not for the expression of pan-neuronal markers.

In summary, all the homologues in the model organisms share conserved zinc-finger motifs as well as functional roles in sensory neuron, noradrenergic neuron, and neuronal precursor cells.

NEUROENDOCRINE-SPECIFIC EXPRESSION PATTERNS OF INSM1

The most unique feature of INSM1 in both mammals and invertebrates is its restricted tissue distribution pattern. INSM1 expression is very low or absent in adult tissues. So far, an INSM1-specific antibody is still lacking to perform a comprehensive immunohistochemical analysis of its protein expression pattern. However, in situ hybridization, RT-PCR, and/or Northern blot analyses have generated some conflicting results about INSM1 expression in adult tissues (23,24,25). An observation in D. melangaster may potentially explain this discrepancy. With the use of in situ hybridization, nerfin-1 expression is detected in many neuronal precursor cells including all delaminating CNS neuroblasts. However, analysis of the nerfin-1 protein expression pattern revealed a more limited expression profile. Nerfin-1 protein was detected in neuronal progenitors that divide just once to produce neurons and then only transiently in nascent neurons. Therefore, to understand this inconsistency between the mRNA and protein expression profiles, the 3′-UTR was analyzed. Multiple conserved recognition sites for 18 different miRNAs were found in the 3′UTR of nerfin-1. Replacement of the nerfin-1 3′-UTR with the SV40 3′-UTR resulted in prolonged reporter protein expression in neurons (18). Therefore, similar post-transcriptional regulation mechanisms in other organisms may be active. Nonetheless, it is clear that INSM1 is detected dominantly in the developing pancreas and nervous system as well as in tumors of neuroendocrine origin (1, 26, 27).

Normal embryonic expression of INSM1

In situ hybridization of mouse and human tissues revealed that the INSM1 transcript was detected in the developing forebrain, midbrain, hindbrain, olfactory epithelium, retina, cerebellum, pancreas, thymus, thyroid, adrenal gland, and endocrine cells of the gastrointestinal tract (23, 27, 28). The expression level of INSM1 is dramatically decreased after birth and diminished completely in adulthood. However, an extensive human brain expression study demonstrated that the INSM1 transcript is transiently expressed in progenitors and nascent neurons (postmitotic neuronal precursors) throughout embryonic and adult neurogenesis (28). For example, in the developing cerebellum, INSM1 is present in proliferating progenitors of the outer external granule layer and in postmitotic cells of the inner external granule layer but not in mature granule cell neurons. In the mammal cortex, basal (intermediate) progenitors are the major source of neurons. However, the molecules required for basal cell specification are largely uncharacterized. INSM1 was identified using gene expression profiling with embryonic day (E) 9.5–11 mouse dorsal telencephalon (29). INSM1 expression peaked during the onset of neurogenesis and was specifically expressed in neuronal progenitor cells (PCs). In situ hybridization showed INSM1 expression in brain areas associated with neurogenesis, including the external granule cell layer of the developing cerebellum, the dentate gyrus of the postnatal hippocampus, and the wall of the lateral ventricle at postnatal day 7 that persists into adulthood. The expression pattern is concentrated specifically in neurogenic PCs rather than newborn neurons. In mice lacking the transcription factor INSM1, the radial thickness of the cortical plate was markedly reduced, and the number of BPs was decreased by half (29). BPs are defined as PCs that divide away from the ventricular surface in the basal region of the ventricular zone and in the adjacent subventricular zone. BPs are more abundant in the telencephalon, the CNS region with the greatest production of neurons. With the use of both the loss-of-function mouse model and a gain-of-function analyses, INSM1 was shown to play a role as the master regulator of BP biogenesis in the neocortex (29). Forced expression of INSM1 in the dTel of E10.5 mice was carried out to substantiate the role of INSM1 in neurogenesis. Forced premature expression of INSM1 resulted in a >2-fold increase in the abundance of basal mitoses. However, apical mitoses were not increased. Functionally, INSM1 null mice revealed a panneurogenic role of INSM1 in neural PCs.

The INSM1 promoter was identified within ~500 bp 5′-upstream of the transcription start site and regulates INSM1 expression in neuroendocrine tumors (30). A transgenic mouse model using a ~1.7 kb 5′-upstream INSM1 promoter sequence linked to a LacZ reporter gene was generated to study the spatial and temporal expression pattern of INSM1. LacZ staining was detected as early as E9.5 to newborn time points with strong LacZ activity in the forebrain, midbrain, hindbrain, spinal cord, retina, lens, trigerminal ganglion, olfactory bulb, and pineal gland (26). The INSM1 promoter-reporter gene marked strong expression patterns in the developing nervous system that correlated with subsequent in situ hybridization studies in humans and mice (28). However, some regions identified by in situ hybridization or Northern blot analyses were not positive in the INSM1 promoter transgenic animals, including the pancreas, thyroid, adrenal gland, and thymus. This discrepancy may be explained by the use of a promoter region (~1.7 kb) lacking key elements required for or simply by the human INSM1 promoter sequence not functioning as anticipated in a mouse animal model. In conclusion, INSM1 expression is predominantly found in developing neuroendocrine tissues and neuronal progenitors in the brain.

INSM1 expression in neuroendocrine tumors

INSM1 was originally isolated from an insulinoma subtraction library (1). Subsequent studies revealed that INSM1 expression is quite specific for neuroendocrine tumors and is absent from other non-neuroendocrine tumors. An extensive neuroendocrine tumor marker analysis in lung cancer demonstrated that 97% (30/31) of small cell lung carcinoma cell lines are positive for INSM1 expression. INSM1 expression also appears in nonsmall cell lung carcinoma with a neuroendocrine phenotype. INSM1 showed a high concordance with the other neuroendocrine markers l-dopa decarboxylase and chromogranin A (31). In addition, several microarray gene expression profiling experiments of small cell lung cancer cells identified INSM1 as a highly specific marker that is abundantly expressed in neuroendocrine lung tumors (32, 33). INSM1 was identified as a prominent differential marker for SCLC along with Hash1 and gastrin-releasing peptide. Using the human 1.7 kb INSM1 promoter region, Pedersen et al. (34) demonstrated the in vitro efficacy and specificity of this promoter to drive the expression of the HSV-tk suicide gene in human SCLC cell lines. Recently, we analyzed the therapeutic potential of an adenoviral INSM1 promoter-driven HSV-tk construct in primitive neuroectodermal tumors (PNETs). Both in vitro multiplicity of infection and ganciclovir sensitivity studies indicated that the INSM1 promoter could mediate specific expression of the HSV-tk gene and selectively kill INSM1 positive PNETs. In vivo intratumoral adenoviral delivery demonstrated that the INSM1 promoter directs the HSV-tk gene expression in a nude mouse tumor model and effectively repressed tumor growth in response to GCV treatment (unpublished results). These studies demonstrate the tremendous potential for targeted cancer gene therapy by taking advantage of a neuroendocrine tumor-specific promoter.

INSM1 is expressed in the majority of neuroendocrine tumors tested so far, including insulinoma, small cell lung carcinoma, pituitary tumor, pheochromocytoma, medullary thyroid carcinoma, medulloblastoma, neuroblastoma, and retinoblastoma (1, 26). The expression profile of INSM1 in both humans and rodents reveals that it is abundant in the fetal brain and developing neuroendocrine system and is silenced or significantly reduced/restricted in adult tissues. However, the strong activation of the INSM1 gene in tumors of neuroendocrine origin suggests a dedifferentiation event occurs in neuroendocrine tumors that mimic normal embryonic development. This information strongly suggests that INSM1 could play a pivotal role during neuroendocrine cell differentiation.

BIOLOGICAL FUNCTIONS OF INSM1 IN NEUROENDOCRINE DIFFERENTIATION

Role of INSM1 in the endocrine pancreas transcriptional network

After determination of the consensus binding site for INSM1, further biochemical analyses showed that INSM1 could bind to multiple target gene promoter sequences and suppress target gene transcription. INSM1 binds to its own promoter, suggesting autoregulation as a self-control feedback mechanism (10). Two target genes, NeuroD/β2 and insulin, were identified to contain a functional INSM1 binding site in their proximal promoter regions. Transient cotransfection reporter gene assays with an INSM1 expression plasmid down-regulated expression of both endogenous target genes. INSM1 protein was localized on the endogenous insulin or NeuroD/β2 promoter sequence by chromatin immunoprecipitation assays (35, 36). The molecular mechanisms underlying the INSM1 transcriptional repressor activity is mediated through the recruitment of cyclin D1 and histone deacetylase-3 (HDAC-3), leading to the modification of the acetylation state of histone H3/4. In vitro induction of AR42J amphicrine cells, pancreatic adenocarcinoma cells (PANC-1), and normal human ductal epithelial cells into insulin-producing cells suggests that INSM1 gene expression is closely associated with the expression of islet-specific transcription factors (23, 24, 37). Biochemical analysis of the INSM1 promoter sequence (−426/+40 bp) revealed that three E-box elements were likely responsible for the tissue-specific promoter activity (26). The basic helix-loop-helix transcription factors NeuroD/β2/E47 and Ngn3/E47 were found to compete or coordinate for binding to the E3-box and strongly activate INSM1 gene expression (26, 37). These data suggest that neurogenin3 (Ngn3) induces both INSM1 and NeuroD/β2 expression. Because Ngn3 is expressed transiently in the endocrine pancreas, NeuroD/β2 maintains INSM1 expression when Ngn3 becomes silenced (23, 37, 38). Since INSM1 negatively regulates NeuroD/β2 and itself, INSM1 most likely plays a role as the checkpoint regulator of NeuroD/β2 and will eventually suppress its own gene expression in adult tissues. Mellitzer et al. (23) analyzed developing pancreas from various mutant animals and clearly showed that INSM1 is an immediate downstream target gene of Ngn3 but that it is situated upstream of the NeuroD/β2, Pax4, Arx, and Pax6 transcription factors during pancreatic endocrine cell differentiation. The global INSM1 mutant mouse model is embryonically lethal (25). In the knockout mouse, endocrine pancreas precursor cell development appears normal; however, β-cell development is severely impaired. With the use of microarray analysis of the INSM1 knockout mouse pancreas tissue, a large number of islet-associated transcripts were down-regulated probably due to the impairment of pancreatic endocrine cell differentiation. Therefore, both in vitro and in vivo data support an essential role for INSM1 in pancreatic endocrine cell development. The involvement of INSM1 in the islet-specific transcriptional network could explain its functional role in β-cell development (Fig. 3).

Figure 3.
INSM1 in endocrine pancreas transcriptional network. Pancreas is derived from the endoderm layer. Multiple transcription factors contribute to the progression of pancreatic endocrine and exocrine cell differentiation. Transcription factors Pdx-1 and PTF1a-p48 ...

Functional role of INSM1 in the SA transcriptional network

The observation of early embryonic lethality and a deficiency of catecholamine synthesis in INSM1 mutant mice indicates that INSM1 could be a crucial component in SA lineage differentiation (39). Mutant mice show a marked change in terminal differentiation of chromaffin cells and reduced expression of genes whose protein products control catecholamine synthesis and secretion. Catecholamines are essential for mouse fetal development and survival (40, 41). SA lineage cells are derived from neural crest (NC) cells, which give rise to sympathetic neurons and adrenal chromaffin cells. NC cells are directed to the SA cell fate by BMPs secreted from the wall of the dorsal aorta. SA precursor cells, sympathetic neurons, and chromaffin cells express common sets of genes essential for their differentiation and catecholamine synthesis. Mash 1, Phox2a/b, Hand2, and Gata2/3 are required for SA lineage formation. INSM1 mutant mice showed a remarkable phenotype similarity to the Mash1 mutant mice, suggesting that INSM1 may mediate aspects of Mash 1 functions in the subtype-specific differentiation of the SA precursors (39). Castro et al. (42) predicted that INSM1 could be a direct target of Mash1 regulation. INSM1 expression is not correctly initiated in the SA lineage of Phox2b and Mash1 mutant mice (39). This result indicates that both Phox2b and Mash 1 are upstream regulators of INSM1 in SA development. Therefore, in the SA lineage transcriptional network, INSM1 is a new player in the control of sympathetic neuron and chromaffin cell differentiation and is located between Mash1/Phox2b and Phox2a/Hand2/Gata3 in the SA transcriptional cascade (Fig. 4).

Figure 4.
INSM1 in SA lineage transcriptional network. SA precursors are generated from NC cells that give rise to mature neurons of secondary sympathetic ganglia, to chromaffin cells of adrenal medullas, and to extra-adrenal chromaffin tissue. In dorsal aorta ...

INSM1 in neuroendocrine signaling pathways

INSM1 is transiently expressed in developing neuroendocrine tissues and is abundantly expressed in a variety of neuroendocrine tumors. Gene deletion analysis indicated that INSM1 plays important roles in pancreatic endocrine differentiation, in SA lineage development, and in the developing mouse neocortex (22, 23, 25, 29, 39). Both upstream and downstream regulatory genes of INSM1 are critical factors involved in these transcriptional specification networks. In addition, synchronized Notch signal inactivation revealed a role for INSM1 in the initial program of PC differentiation in retinal neurons (43). This result is consistent with the role of the Notch signaling pathway in antagonizing Ngn3 during pancreatic endocrine differentiation and Xash1 in noradenergic neuron progenitor differentiation (22, 44). INSM1 is strongly expressed in the developing cerebellum and medulloblastoma cells (45). Medulloblastoma is a pediatric brain tumor arising from the cerebellar granule layer and has been linked to the aberrant activation of the sonic hedgehog (Shh) pathway (46, 47). Similarly, development of the cerebellum is largely dependent on Shh signaling. During cerebellar development in mice, INSM1 was identified as one of the genes specifically up-regulated in the time window in which the Shh pathway is strongly activated. Moreover, INSM1 expression could be induced in cultured cerebellar granule precursors cells treated with Shh, providing a direct link of INSM1 to the Shh signaling pathway in cerebellum development and medullablastoma tumors (45).

A yeast two-hybrid screen for INSM1-interacting proteins revealed several interesting cellular factors that connect INSM1 to various signaling pathways. An important prime candidate target is the cell cycle regulator cyclin D1. Experimental evidence showed that cyclin D1 bound to INSM1 directly and facilitated HDAC-3 recruitment, which subsequently deacetylates histone H3/H4 to promote transcriptional repression (35, 36). However, cyclin D1 binding to INSM1 demonstrated a dual functional role in both cell cycle dependent and independent pathways (35). The INSM1 binding site on the cyclin D1 protein is located at the cyclin box, the same binding region for cyclin-dependent kinase 4 (CDK4) (48). CDK4 binding to cyclin D1 is essential for initiation of cell cycle signaling through the retinoblastoma (Rb) protein. When Rb is phosphorylated, it releases E2F, which results in gene activation. Our recent study (49) shows that binding of INSM1 to cyclin D1 disrupts CDK4 binding, induces Rb protein hypophosphorylation, and causes cell cycle arrest in non-neuroendocrine cells. Therefore, we hypothesize that transient expression of INSM1 at the appropriate developmental window functions as a differentiation factor by facilitating cell cycle exit and entrance into normal neuroendocrine cell differentiation (Fig. 5). Recent studies (29) in the developing mouse telencephalon support this hypothesis, since forced overexpression of INSM1 in NE cells of E10.5 mouse brain led to a 20% decrease of bromodeoxyuridine-incorporated cells present in the ventricular zone. This reduction represents a lengthening of cell cycle rather than cell cycle withdrawal. Further, this mechanism seems to be conserved evolutionarily, since a report (21) using medaka observed that forced expression of INSM1 in non-neuronal cells slowed down the cell cycle without triggering apoptosis.

Figure 5.
INSM1 displays both cell cycle-dependent and -independent functions. A) INSM1 interacts with cyclin D1 through the cyclin box binding region, which disrupts CDK4 binding and subsequently inhibits Rb protein phosphorylation, E2F release, gene activation, ...

CONCLUSION AND PERSPECTIVES

Neuroendocrine differentiation controls proper formation of the hormone-producing endocrine cells that modulate the body’s metabolism in multiple organs and neuroendocrine systems. In this review, we report a unique neuroendocrine-specific transcription factor, INSM1. INSM1 (or IA-1) was originally isolated from a human insulinoma subtraction library (1). The deduced amino acid sequence predicted a zinc-finger DNA-binding protein. The C-terminal portion of the INSM1 protein binds to a consensus sequence (TG/TC/TC/TT/AGGGGG/TCG/A), which leads to the identification of several target genes containing the INSM1-binding site in their promoter regions. Biochemical studies revealed that INSM1 is a transcriptional repressor. It is functionally active to suppress NeuroD/β2, insulin, and its own expression in pancreatic islets. The neurogenic transcription factors Ngn3 and Mash1 serve as upstream positive regulators of INSM1 expression in pancreatic and SA cells, respectively. INSM1 mutant mice demonstrate multiple phenotypes, including impairment of pancreatic islet cell development, catecholamine synthesis deficiency and secretion, and reduced neocortex radial cortical thickness due to a reduction in basal PCs (25, 29, 39). Catecholamines are essential for fetal mouse development and are the main reason that INSM1 mutant mice die in utero. Due to the early embryonic lethality of the global knockout, conditional targeted animals are needed to dissect the individual effect of INSM1 in each endocrine organ and/or neuroendocrine system. Furthermore, INSM1 is abundantly expressed in tumors of neuroendocrine origin. For example, INSM1 is expressed in almost 100% of small cell lung carcinomas (SCLC) but rarely in normal adult lung tissues. It is tempting to speculate that INSM1 expression in SCLC could contribute to the unique characteristics of SCLC. Since SCLC responds to chemotherapy and radiation therapy more effectively than other kinds of lung cancer (50), INSM1 expression could be used as both an neuroendocrine marker and a prognostic marker for therapeutic strategies (31). We have discussed and tested the potential usefulness of INSM1 promoter for targeted cancer gene therapy (34). It is conceivable that the more we learn about how INSM1 functions in neuroendocrine differentiation, the better we can design strategies to control the proper synthesis and expression of important hormones in the neuroendocrine system. INSM1 represents a newly identified factor that plays a critical role during both endocrine and neuronal cell differentiation.

Acknowledgments

This work was supported by funds from the Research Institute for Children, Children’s Hospital, New Orleans; a grant from the Diana Helis Henry Medical Research Foundation (to M.B.B.); and a grant from the National Institute of Diabetes and Digestive and Kidney Diseases (DK-61436; to M.S.L.).

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