Id1 mRNA Levels Research Articles

Id1 Regulates the Choice of Hematopoietic Stem Cells to Self-Renew or Commit to Differentiation.

Id proteins belong to the basic helix-loop-helix family of transcription factors and act as dominant negative forms of E-protein transcriptional activators. Id mediated E protein silencing has an essential role in restricting differentiation and maintaining self-renewal in embryonic stem cells. However, the role of Id1 in adult stem cells including HSCs has not been described thus far. Having detected relatively high levels of Id1 mRNA in murine adult HSCs (compared to the committed myeloid progenitor cells) we examined the in vivo HSC function in Id1 deficient mice.We observed a >2 fold reduction in HSC frequency in the bone marrow in 8-10w old Id1−/− mice compared to Id1+/+ animals, detected by both lin-c-kit+Sca-1+ (LKS) cell surface marker profile and Hoechst 33342 dye efflux - “side population” phenotype, as well as a ~25% decrease in total bone marrow cellularity. Although Id1 deficient HSCs show robust long-term competitive repopulating capacity in primary transplant recipients, they have markedly impaired hematopoietic function upon secondary transplantation. Id1 null HSCs show a higher rate of S-phase entry in vivo as measured by 3 day BrdU incorporation (ko: 79.0±3.9% vs. wt: 49.7±7.4) and faster initial doubling times in response to cytokine stimulation in vitro during the first 2 days of culture. This failure to maintain normal HSC numbers and the diminished repopulating capacity, in the presence of enhanced cell cycling, suggests a defect in the regulation of self-renewal in Id1 deficient HSCs. Considering the general function of Id1 as an inhibitor of differentiation, the observed effect of Id1 loss could be explained by the excessive recruitment of LKS cells into the actively proliferating differentiated progenitor pool, at the expense of their self-renewal capacity. Consistent with this, sorted Id1−/− HSCs show accelerated expression of cell surface lineage markers in vitro and an increased ratio of CFU-S8 /CFU-S12 in the in vivo spleen colony forming assay.Global gene expression profiling of Id1+/+ vs. Id1−/− hematopoietic cells (using Affymetrix MOE430 Plus chips) revealed insignificant transcriptional deregulation in the committed myeloid progenitor subsets (CMP, GMP, MEP) in the absence of Id1. Meanwhile, Id1−/− HSCs showed a marked change in gene expression pattern (more than 1500 genes with a ≥2 fold difference in expression levels). Differentially regulated transcripts in Id1+/+ vs. Id1−/− HSCs significantly overlap (~30%) with the observed changes in gene expression that accompany the transition of HSCs to the common myeloid progenitor phenotype. Specifically, genes such as c/EBPα and GATA1 are significantly upregulated in Id1 null immunophenotypic HSCs, consistent with an earlier than normal commitment to myelo-erythroid differentiation. In contrast, several known transcriptional regulators of HSC self-renewal (Bmi1, Gfi1, HoxB4) show no significant change in expression pattern. These data clearly indicate the unique role of Id1 in regulating HSC self-renewal by restricting the rate of HSC commitment to the myeloid progenitor cell fate.

Ubiquitin-Proteasome-mediated Degradation, Intracellular Localization, and Protein Synthesis of MyoD and Id1 during Muscle Differentiation

Mammalian skeletal myogenesis results in the differentiation of myoblasts to mature syncytial myotubes, a process regulated by an intricate genetic network of at least three protein families: muscle regulatory factors, E proteins, and Id proteins. MyoD, a key muscle regulatory factor, and its negative regulator Id1 have both been shown to be degraded by the ubiquitin-proteasome system. Using C2C12 cells and confocal fluorescence microscopy, we showed that MyoD and Id1 co-localize within the nucleus in proliferating myoblasts. In mature myotubes, in contrast, they reside in distinctive subcellular compartments, with MyoD within the nucleus and Id1 exclusively in the cytoplasm. Cellular abundance of Id1 was markedly diminished from the very onset of muscle differentiation, whereas MyoD abundance was reduced to a much lesser extent and only at the later stages of differentiation. These reductions in MyoD and Id1 protein levels seem to result from a change in the rate of protein synthesis rather than the rate of degradation. In vivo protein stability studies revealed that the rates of ubiquitin-proteasome-mediated MyoD and Id1 degradation are independent of myogenic differentiation state. Id1 and MyoD were both rapidly degraded, each with a t 1/2 approximately = 1 h in myoblasts and in myotubes. Furthermore, relative protein synthesis rates for MyoD and Id1 were significantly diminished during myoblast to myotube differentiation. These results provide insight as to the interaction between MyoD and Id1 in the process of muscle differentiation and have implications for the involvement of the ubiquitin-proteasome-mediated protein degradation and protein synthesis in muscle differentiation and metabolism under abnormal and pathological conditions.

Denervation stimulates apoptosis but not Id2 expression in hindlimb muscles of aged rats.

Inhibitors of differentiation (Id) proteins are repressors of myogenic regulatory factors and have been implicated in apoptosis and muscle atrophy during aging. Indeed, we have previously found that Id levels are elevated in muscles from old rodents, possibly as a consequence of loss of alpha-motoneurons during senescence. To determine if Id2 proteins increase after denervation and if this is accompanied by increased apoptosis in aged as compared with adult animals, the gastrocnemius and soleus muscles were denervated in 1 limb of Fischer 344 x Brown Norway rats aged 9 months (adult, n = 12) and 33 months (aged, n = 9), while the contralateral limb served as the intra-animal control. After 14 days, the muscles in each limb were removed. The levels of Id1, Id2, and Id3 mRNA and protein were significantly greater in muscles of old as compared with young adult rats. Denervation, however, did not significantly increase Id1, Id2, and Id3 mRNA in soleus or gastrocnemius muscles from either young or old rats. Also Id2 protein levels were similar in denervated and control muscles from young adult and old rats. In young adult rats only, denervation induced an increase in Id1 and Id3 protein levels in both the soleus (Id1 113%; Id3 900%) and gastrocnemius (Id1 86%; Id3 80%). Denervation induced a significant increase in caspase 8 in both soleus and gastrocnemius muscles from young (101% and 147%, respectively) and old rats (167% and 190%, respectively). Bax protein levels, as estimated by western blots, increased by 726% and 1087% after denervation in the soleus and by 368% and 49% in the gastrocnemius muscles of young and old rats, respectively. The data suggest that the denervation-induced muscle loss was at least partly due to apoptosis as indicated by elevated caspase 8 and Bax levels in denervated muscles. While Id2 may have a role in aging-induced sarcopenia, Id2 does not appear to directly regulate apoptosis during denervation. The elevated Id expression in muscles from aged animals is therefore not a direct consequence of loss of alpha-motoneurons during senescence.

Down-regulation of Id-1 expression is associated with TGFβ1-induced growth arrest in prostate epithelial cells

Transforming growth factor β1 (TGFβ1) plays important roles in the regulation of cell growth and differentiation in both normal and malignant prostate epithelial cells. Although certain pathways have been suggested, the mechanisms responsible for the action of TGFβ1 are not well understood. In the present study, using a human papilloma virus 16 E6/E7 immortalized prostate epithelial cell line, HPr-1, we report that TGFβ1 was able to suppress the expression of Id-1, a helix–loop–helix (HLH) protein, which plays important roles in the inhibition of cell differentiation and growth arrest. In addition, a decrease at both Id-1 mRNA and protein expression levels was associated with TGFβ1-induced growth arrest and differentiation, indicating that Id-1 may be involved in TGFβ1 signaling pathway. The fact that up-regulation of p21 WAF1, one of the downstream effectors of Id-1, was observed after exposure to TGFβ1 further indicates the involvement of Id-1 in the TGFβ1-induced growth arrest in HPr-1 cells. However, increased expression of p27 KIP1 was also observed in the TGFβ1-treated cells, suggesting that in addition to down-regulation of Id-1, other factors may be involved in the TGFβ1-induced cell growth arrest and differentiation in prostate epithelial cells. Our results provide evidence for the first time that TGFβ1 may be one of the upstream regulators of Id-1.

CDNA array analysis of pineal gene expression reveals circadian rhythmicity of the dominant negative helix-loop-helix protein-encoding gene, Id-1.

The pineal gland is a major output of the endogenous vertebrate circadian clock, with melatonin serving as the output signal. In many species, elevated nocturnal melatonin production is associated with changes in pineal gene expression. In the current study, cDNA array analysis was used in an attempt to identify additional genes that exhibit day/night differential expression in the rat pineal gland. This revealed 38 candidate genes, including Id-1 (inhibitor of DNA binding and differentiation). Id-1 encodes a helix-loop-helix (HLH) protein that lacks a basic DNA binding domain and could affect pineal physiology via a dominant negative trans-acting regulatory activity. For this reason Id-1 was selected for further analysis. Id-1 was expressed in a major population of pineal cells and the Id-1 protein was associated with a nuclear complex. The levels of Id-1 mRNA and protein exhibit approximately six-fold day/night rhythms. In contrast, the related genes Id-2 and Id-3 do not exhibit marked day/night differences in pineal expression. Rhythmic Id-1 expression is primarily limited to a C-terminally extended splice variant of Id-1, which would restrict the functional output of the rhythm to protein binding partners of this isoform of Id-1. Our findings add to the body of evidence indicating that transcriptional regulators play a role in neuroendocrine rhythms, and extend this by introducing the concept of a dominant negative HLH involvement. The rhythm in Id-1 in the pineal gland provides an experimental opportunity to identify Id-1-binding partners which may also be involved in Id-1 activity in other functional contexts.

Tumor necrosis factor-alpha regulation of the Id gene family in astrocytes and microglia during CNS inflammatory injury.

The inhibitors of DNA binding (Id) gene family is highly expressed during embryogenesis and throughout adulthood in the rat central nervous system (CNS). In vitro studies suggest that the Id gene family is involved in the regulation of cell proliferation and differentiation. Recently, Id gene expression was shown to be expressed in immature and mature astrocytes during development and upregulated in reactive astrocytes after spinal cord injury. These results suggest that the Id gene family may play an important role in regulating astrocyte development and reactivity; however, the factors regulating Id expression in astrocytes remain undefined. Tumor necrosis factor-alpha (TNF alpha), a proinflammatory cytokine, is thought to play a crucial role in astrocyte/microglia activation after injury to the CNS. To determine if TNF alpha plays a role in Id gene expression, we exogenously administered TNF alpha into developing postnatal rats. We report that TNF alpha injections resulted in a rapid and transient increase in both cell number and mRNA expression for Id2 and Id3 when compared to levels observed in noninjected or control-injected animals. Id1 mRNA levels were also upregulated after TNF alpha treatment, but to a lesser degree. Significant increases in TNF alpha-induced Id2 and Id3 mRNA were observed in the ventricular/subventricular zone, cingulum and corpus callosum. TNF alpha also increased Id2 mRNA expression in the caudate putamen and hippocampus at the injection site. Id2 and Id3 mRNA+ cells were identified as GFAP+ and S100 alpha + astrocytes as well as ED1+ microglia. This is the first report to show TNF-alpha-induced modulation of the Id gene family and suggests that Id may be involved in the formation of reactive astrocytes and activated microglia in the rodent brain. These results suggest a putative role for the Id family in the molecular mechanisms regulating cellular responsiveness to TNF alpha and CNS inflammation.

Injury selectively down-regulates the gene encoding for the Id4 transcription factor in primary cultures of forebrain astrocytes.

Astrogliosis is an important component of the response to injury of the central nervous system (CNS). The Id family of helix-loop-helix (HLH) transcription factors has been implicated in the regulation of cellular differentiation in several different lineages and may contribute to the regulation of astrogliosis. We examined the expression of Id genes in primary cultures of mouse forebrain astrocytes under experimental conditions in which astrogliosis was elicited by mechanical injury. Astrocyte cultures expressed the four known members of the Id gene family, Id1, Id2, Id3, and Id4. After injury, at a time when astrocytes developed the characteristic phenotypic changes of astrogliosis, Id4 expression decreased dramatically. Id1, Id2, and Id3 mRNA levels did not change. These results identify Id4 as a candidate marker of astroglial activation in culture and suggest that Id4 expression plays a role in the process of astrogliosis.

Expression of Id-1 mRNA and Protein in the Post-Ischemic Regenerating Rat Kidney

The basic helix-loop-helix (bHLH) class of proteins are of major importance in controlling tissue-specific gene expression. The actions of the bHLH proteins are inhibited by a related class of proteins, inhibitors of differentiation (Id). We have studied the expression of one of these latter proteins, Id-1, in the normal and post-ischemic regenerating rat kidney by immunocytochemistry, Western blot and RNase protection assay (RPA) and correlated Id-1 regulation to the expression of vimentin and proliferating cellular nuclear antigen (PCNA). In the normal kidney strong immunostaining for Id-1 was found in the distal nephron, especially in the distal convoluted tubule in the cortex. In particular, the perinuclear region was intensely stained in the cells of the distal tubule. mRNA for Id-1 was detectable by RPA on total RNA extracted from the renal cortex of sham-operated animals. The Id-1 monomer was detected on Western blots of normal animals. Vimentin was expressed in the mesangial cells of the glomeruli and in cells in the interstitium while tubule cells were negative. The labeling intensity for PCNA was low in all cellular compartments in the normal kidney. In the regenerating kidneys at various time intervals, the expression of Id-1-like immunoreactivity was widespread in the regenerating dedifferentiated tubule cells while by the end of the study period, more highly differentiated tubule cells appeared to lose their staining. On Western blots the Id-1 monomer was undetectable and instead strong staining was seen in the high molecular range. Id-1 mRNA levels in the regenerating kidneys did not differ significantly when compared to sham. PCNA labeling was intense in the regenerating kidneys at all time periods studied, indicating the intense proliferative activity in the regenerating kidneys. Vimentin expression in the renal tubule cells was increased from day 3 and onward. The data are consistent with a hypothesis in which Id-1 regulates differentiation of renal tubule cells in the post-ischemic regenerating rat kidney.

Expression of the helix-loop-helix genes Id-1 and NSCL-1 during cerebellar development.

Neurons throughout the central nervous system (CNS) undergo proliferation, migration, and differentiation during their histogenesis. Although numerous regulatory molecules are expressed in developing neurons, it is unknown whether most of these molecules have the same function throughout the CNS or play different roles in different neuronal populations. Previous studies have shown that Id-1 and NSCL-1 are expressed at high levels in the ventricular and subependymal zones, respectively, of the embryonic brain. In the present study, the expression of Id-1 and NSCL-1 was further investigated during postnatal development of the cerebellum. By Northern blot hybridization analysis, the expression levels of Id-1 and NSCL-1 mRNA were developmentally regulated in the cerebellum, with the highest mRNA levels coinciding with the time of maximal granule cell histogenesis. By in situ hybridization, NSCL-1 mRNA was found in the premigratory zone of the external granule layer (EGL), a structure developmentally analogous to the subependymal zone of the embryonic brain. In normal mice, Id-1 mRNA was found to be transiently expressed in the upper internal granule layer (IGL), a population of cells that recently completed their migration from the EGL. In the mouse mutant weaver, Id mRNA was only seen in granule cells that have reached their normal positions in the IGL. No Id-1 hybridization signal was observed in the large numbers of granule cells remaining in the EGL of weaver mice, indicating that Id-1 expression is controlled by spatial cues. The lack of Id-1 expression in ectopic weaver granule cells is compatible with previous suggestions of arrested differentiation. These results support the idea that transcriptional regulators of the helix-loop-helix gene family play important roles in neuronal development, exhibiting region-specific expression and function.

Regulation of rat liver type 1 iodothyronine deiodinase mRNA levels by testosterone

To investigate the underlying mechanisms of sex-related differences in liver type 1 iodothyronine deiodinase (ID1), we studied the sex-related differences and roles of sex steroids in liver ID1 mRNA levels in the rat. In both euthyroid and thyroidectomized rats, liver ID1 activity and ID1 mRNA levels in female rats were less than those in male rats. A positive correlation was observed between liver ID1 activity and ID1 mRNA levels. Liver ID1 activity and ID1 mRNA levels in male rats decreased after orchiectomy, and were increased to control levels by testosterone administration. Ovariectomy or β-estradiol administration did not alter liver ID1 activity or ID1 mRNA levels in female rats. ID1 mRNA levels in cultured rat hapatocytes were significantly increased by testosterone, but not by β-estradiol. These results suggest that the sex-related differences in liver ID1 activity are attributable to differences in ID1 mRNA levels, and that testosterone plays an important role in the sex-related differences in liver ID1 mRNA levels.

Id gene expression during development and molecular cloning of the human Id-1 gene

Id genes encode helix-loop-helix proteins that inhibit transcription by forming inactive heterodimers with basic helix-loop-helix (bHLH) proteins. bHLH proteins normally form either homodimers or heterodimers with other bHLH proteins and bind to a DNA sequence element activating transcription. Id-containing heterodimers are inactive because Id proteins lack the basic amino acid region necessary to form a DNA-binding domain. We have examined the relative levels of Id-1 and Id-2 mRNA during normal development and in malignant tissues. In the course of these experiments we cloned and sequenced the human Id-1 cDNA. Two related cDNA molecules encoding human Id-1 mRNAs were identified. Id-1a is a cDNA of 958 nucleotides and can encode a protein of 135 amino acids. Id-1b cDNA is 1145 nucleotides, can encode a protein of 149 amino acids, and appears to be a splice variant of Id-1a. The amino acid sequence of human Id-1 is greater than 90% homologous to that of mouse Id-1. The patterns of Id-1 and Id-2 expression during mouse development vary widely, and we detected Id-1 expression in human fetal and adult tissues from lung, liver, and brain. High Id-1 mRNA expression was found in many human tumor cell lines, including those isolated from nervous system tumors. We mapped Id-2 to human chromosome 2p25.

Expression and differential regulation of Id1, a dominant negative regulator of basic helix-loop-helix transcription factors, in glomerular mesangial cells.

Id is a family of dominant negative helix-loop-helix (HLH) proteins that block cell-specific transcription mediated by basic HLH (bHLH) transcription mediated by basic HLH (bHLH) transcription factors. We have analyzed Id1 expression in mesangial cells as a first step towards understanding the putative role of bHLH transcription factors in cell type-specific gene expression in the kidney. Glomerular mesangial cells expressed an abundant 1.1 kb mRNA transcript for Id1, but in contrast to other cell types Id1 mRNA was expressed in both randomly cycling cells and in serum-deprived, quiescent cultures. When quiescent mesangial cells were treated with serum to re-enter G1, Id1 mRNA levels were rapidly (2-4 h) and transiently down-regulated. Down-regulation of Id1 mRNA following addition of serum to mesangial cells was cell type-specific and contrasted with induction of Id1 by serum in BHK-21 and 3T3 fibroblasts. Down-regulation of Id1 mRNA correlated with mitogenesis and occurred when quiescent cells were treated with growth factors that activate G protein-coupled receptors and receptor protein tyrosine kinases but not with a non-mitogenic cAMP analog. Down-regulation of Id1 by growth factors required de novo protein synthesis, suggesting that a labile protein was involved. Appearance of E-box DNA binding activity in mesangial cell extracts followed down-regulation of Id1 message. Steady state Id1 mRNA levels and E-box DNA binding activity were not tightly correlated, suggesting complex regulation of Id1 activity. mRNA transcripts for E2A gene products were also expressed in mesangial cells, but these cells failed to express mRNAs for MyoA/MyoD-related genes. Collectively, these data demonstrate that Id1 is expressed in renal mesangial cells and suggest that bHLH complexes might be important for transcriptional regulation in the kidney. In addition, the observation that Id1 mRNA is transiently down-regulated by serum in mesangial cells suggests that Id1 gene expression is more complicated than previously appreciated and is tightly regulated in a cell-specific manner.

In vitro effect of rIL-1 & rIL-6 on corticosterone release by isolated rat adrenal cells

To investigate the underlying mechanisms of sex-related differences in liver type 1 iodothyronine deiodinase (ID1), we studied the sex-related differences and roles of sex steroids in liver ID1 mRNA levels in the rat. In both euthyroid and thyroidectomized rats, liver ID1 activity and ID1 mRNA levels in female rats were less than those in male rats. A positive correlation was observed between liver ID1 activity and ID1 mRNA levels. Liver ID1 activity and ID1 mRNA levels in male rats decreased after orchiectomy, and were increased to control levels by testosterone administration. Ovariectomy or β-estradiol administration did not alter liver ID1 activity or ID1 mRNA levels in female rats. ID1 mRNA levels in cultured rat hapatocytes were significantly increased by testosterone, but not by β-estradiol. These results suggest that the sex-related differences in liver ID1 activity are attributable to differences in ID1 mRNA levels, and that testosterone plays an important role in the sex-related differences in liver ID1 mRNA levels.