CREM
cAMP-responsive element modulator (Inducible cAMP early repressor) (ICER)
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Ageing in men is believed to be associated with fertility decline and elevated risk of congenital disorders for the offspring. The previous studies also reported reduced germ and Sertoli cell numbers in older men. However, it is not clear whether ageing in men with normal spermatogenesis affects the testis and germ cell population dynamics in a way sufficient for transmitting adverse age effects to the offspring. We examined men with normal spermatogenesis at different ages concerning effects on persisting testicular cell types, that is the germ line and Sertoli cells, as these cell populations are prone to be exposed to age effects. Ageing was assessed in testicular biopsies of 32 patients assigned to three age groups: (i) 28.8 ± 2.7 years; (ii) 48.1 ± 1 years; and (iii) 70.9 ± 6.2 years, n = 8 each, with normal spermatogenesis according to the Bergmann-Kliesch score, and in a group of meiotic arrest patients (29.9 ± 3.8 years, n = 8) to decipher potential links between different germ cell types. Besides morphometry of seminiferous tubules and Sertoli cell nuclei, we investigated spermatogenic output/efficiency, and dynamics of spermatogonial populations via immunohistochemistry for MAGE A4, PCNA, CREM and quantified A-pale/A-dark spermatogonia. We found a constant spermatogenic output (CREM-positive round spermatids) in all age groups studied. In men beyond their mid-40s (group 2), we detected increased nuclear and nucleolar size in Sertoli cells, indirectly indicating an elevated protein turnover. From the 7th decade (group 3) of life onwards, testes showed increased proliferation of undifferentiated spermatogonia, decreased spermatogenic efficiency and elevated numbers of proliferating A-dark spermatogonia. Maintaining normal sperm output seems to be an intrinsic determinant of spermatogenesis. Ageing appears to affect this output and might provoke compensatory proliferation increase in A spermatogonia which, in turn, might hamper germ cell integrity.
MeSH Terms
- Adult
- Aged
- Aging
- Congenital Abnormalities
- Genetic Diseases, Inborn
- Humans
- Male
- Middle Aged
- Seminiferous Tubules
- Sertoli Cells
- Spermatogenesis
- Spermatogonia
- Spermatozoa
Keywords
- A-dark
- Sertoli cell nuclei
- ageing
- proliferation
- spermatogenesis
- spermatogonia
Despite extensive insights on the interaction between hematopoietic stem cells (HSCs) and the supporting bone marrow (BM) stroma in hematopoietic homeostasis there remains unanswered questions on HSC regulation. We report on the mechanism by which HSCs attain cycling quiescence by addressing a role for inducible cyclic AMP early repressor (ICER). ICER negatively transcriptional regulators of cAMP activators such as CREM and CREB. These activators can be induced by hematopoietic stimulators such as cytokines. We isolated subsets of hematopoietic cells from ten healthy donors: CD34 CD38 /c-kit (primitive progenitor), CD34 CD38 /c-kit (mature progenitor) and CD34 CD38 /c-kit (differentiated lineage-). The relative maturity of the progenitors were verified in long-term culture initiating assay. Immunoprecipitation indicated the highest level of ICER in the nuclear extracts of CD34 /CD38 cells. Phospho (p)-CREM was also present suggesting a balance between ICER and p-CREM in HSC. ICER seems to be responsible for decrease in G1 transition, based on reduced Cdk4 protein, decreased proliferation and functional studies with propidium iodide. There were no marked changes in the cycling inhibitors, p15 and p-Rb, suggesting that ICER may act independently of other cycling inhibitors. The major effects of ICER were validated with BM mononuclear cells (BMNCs) in which ICER was ectopically expressed, and with BMNCs resistant to 5-fluorouracil- or cyclophosphamide. In total, this study ascribes a novel role for ICER in G1 checkpoint regulation in HSCs. These findings are relevant to gene therapy that require engineering of HSCs, age-related disorders that are associated with hematopoietic dysfunction and other hematological disorders.
MeSH Terms
- ADP-ribosyl Cyclase 1
- Aging
- Antigens, CD34
- Blotting, Western
- Cell Cycle
- Cell Differentiation
- Cells, Cultured
- Cyclic AMP Response Element Modulator
- Cyclophosphamide
- Fluorouracil
- Gene Expression
- Hematopoietic Stem Cells
- Humans
- Immunosuppressive Agents
- Proto-Oncogene Proteins c-kit
- Signal Transduction
Keywords
- Bone marrow
- Cell cycle
- Cytokines
- Hematopoietic stem cell
- Inducible cAMP early repressor
- Transcription factors
Human cyclic AMP response modulator proteins (CREMs) are encoded by the CREM gene, which generates 30 or more different CREM protein isoforms. They are members of the leucine zipper protein superfamily of nuclear transcription factors. CREM proteins are known to be implicated in a plethora of important cellular processes within the CNS. Amazingly, little is known about their cellular and regional distribution in the brain, however. Therefore, we studied by means of immunohistochemistry and Western blotting the expression patterns of CREM in developing and adult human brain, as well as in brains of Alzheimer's disease patients. CREM immunoreactivity was found to be widely but unevenly distributed in the adult human brain. Its localization was confined to neurons. In immature human brains, CREM multiple neuroblasts and radial glia cells expressed CREM. In Alzheimer's brain, we found an increased cellular expression of CREM in dentate gyrus neurons as compared to controls. We discuss our results with regard to the putative roles of CREM in brain development and in cognition.
MeSH Terms
- Aged
- Aged, 80 and over
- Aging
- Alzheimer Disease
- Animals
- Cyclic AMP Response Element Modulator
- Dentate Gyrus
- Female
- Humans
- Immunohistochemistry
- Male
- Middle Aged
- Neurons
- Rats
- Rats, Sprague-Dawley
The mechanisms through which estradiol (E2) regulates gonadotropin-releasing hormone (GnRH) neurons to control fertility are unclear. Previous studies have demonstrated that E2 rapidly phosphorylates cAMP response element-binding protein (CREB) in GnRH neurons in vivo. In the present study, we used GnRH neuron-specific CREB-deleted mutant mice [GnRH-CREB knock-outs (KOs)] with and without global cAMP response element modulator (CREM) deletion (global-CREM KOs) to investigate the role of CREB in estrogen negative feedback on GnRH neurons. Evaluation of GnRH-CREB KO mice with and without global CREM deletion revealed normal puberty onset. Although estrus cycle length in adults was the same in controls and knock-out mice, cycles in mutant mice consisted of significantly longer periods of diestrus and less estrus. In GnRH-CREB KO mice, basal levels of luteinizing hormone (LH) and the postovariectomy increment in LH were normal, but the ability of E2 to rapidly suppress LH was significantly blunted. In contrast, basal and postovariectomy LH levels were abnormal in GnRH-CREB KO/global-CREM KO mice. Fecundity studies showed that GnRH-CREB KO with and without global CREM deletion were normal up to ∼9 months of age, at which time they became prematurely reproductively senescent. Morphological analysis of GnRH neurons revealed a significant reduction (p < 0.01) in GnRH somatic spine density of GnRH-CREB KO mice compared to control females. These observations implicate CREB within the GnRH neuron as an important target for E2's negative feedback actions. They also indicate that the rapid modulation of CREB by E2 is of physiological significance in the CNS.
MeSH Terms
- Aging
- Analysis of Variance
- Animals
- CREB-Binding Protein
- Cyclic AMP Response Element Modulator
- Dendritic Spines
- Estradiol
- Estrogens
- Estrous Cycle
- Feedback, Physiological
- Female
- Fertility
- Gene Expression Regulation
- Gonadotropin-Releasing Hormone
- Hypothalamus
- Luteinizing Hormone
- Mice
- Mice, Inbred C57BL
- Mice, Knockout
- Mutation
- Neurons
- Ovariectomy
- Radioimmunoassay
Activation of gene expression by the cAMP-dependent signaling pathway is regulated by members of the cAMP response element binding protein (CREB) family consisting of CREB, CREM, and ATF-1. It is decisively for the understanding of the heart function as to which type of heart cells expresses CREB and/or CREM. Ventricular myocytes and fibroblasts of young (3 months) and old (24 months) rat hearts were separately investigated to analyse CREB, CREM, and phospho-CREB. Western blot showed CREB expression exclusively in fibroblasts but CREM was predominantly detected in ventricular myocytes. CREB-positive nuclei in heart sections were only revealed in fibroblasts. CREB was activated by forskolin (10 microM), PMA (500 nM), and cyclical mechanical strain (1 Hz, 5% elongation) in fibroblasts. The number of CREB-positive myocytes in old rats was larger than in young rats. But CREB could not be activated by forskolin (10 microM) in all myocytes. Our results suggest that the expression of CREB depends on the cell type and the age of the animal. We discuss that modulation of gene expression as it occurs with a age could be affected by the change within the CREB family members.
MeSH Terms
- Aging
- Animals
- Cells, Cultured
- Cyclic AMP Response Element Modulator
- Cyclic AMP Response Element-Binding Protein
- DNA-Binding Proteins
- Fibroblasts
- Heart Ventricles
- Myocytes, Cardiac
- Rats
- Tissue Distribution
- Transcription Factors
Somatostatin is one of the numerous peptides described in the Harderian gland of different animals. With the aim of trying to elucidate its physiological role, we investigated whether this peptide is expressed in OFA rat Harderian gland at different ages and seasons and, if so, studied the regulatory proteins involved in the activation of the somatostatin gene, and also whether it contains any somatostatin receptors. Nursing (4-15-day-old), prepubertal (21-30-day-old), and adult (54-day-old) OFA rats were sacrificed by decapitation throughout the year, and the Harderian glands were excised and immediately frozen in liquid N2. The expression of somatostatin and its receptors was investigated using RT-PCR techniques; additionally, the existence of proteins which bind to cAMP responsive elements (CRE) was investigated using a band-shift technique. The somatostatin gene was expressed in the Harderian gland of rats aged 4-30 days in autumn and winter but not in spring and summer or in older animals. However, the somatostatin receptor was expressed throughout the year at all the ages studied. In the autumn, nuclear proteins binding to CRE (CREB) were present in 8-10-day-old rats but not in younger 4-day-old animals. We conclude that rat Harderian gland cells transcribe the somatostatin gene depending on the season and age of the animals, while its receptor is always present at all the ages studied; the CREB found produces the same retardation complex as ICER (inducible cAMP early repressor), an isoform of CREM (cAMP responsive element modulator), which in the pineal has been shown to be under adrenergic control. Since somatostatin expression is regulated by cAMP mechanisms, it is feasible that the existence of this repressor ICER could explain why somatostatin expression disappears in adult animals once maturation is complete.
MeSH Terms
- Aging
- Animals
- Cyclic AMP Response Element Modulator
- DNA-Binding Proteins
- Female
- Gene Expression Regulation, Developmental
- Harderian Gland
- Male
- Polymerase Chain Reaction
- Rats
- Receptors, Somatostatin
- Repressor Proteins
- Seasons
- Somatostatin
Mammalian spermatogenesis consists of a series of complex developmental processes controlled by the pituitary-hypothalamic axis. This flow of biochemical information is directly regulated by the adenylate cyclase signal transduction pathway. We have previously described the CREM (cyclic AMP-responsive element modulator) gene which generates, by cell-specific splicing, alternative antagonists of the cAMP transcriptional response. Here we report the expression of a novel CREM isoform (CREM tau) in adult testis. CREM tau differs from the previously characterized CREM antagonists by the coordinate insertion of two glutamine-rich domains that confer transcriptional activation function. During spermatogenesis there was an abrupt switch in CREM expression. In premeiotic germ cells CREM is expressed at low amounts in the antagonist form. Subsequently, from the pachytene spermatocyte stage onwards, a splicing event generates exclusively the CREM tau activator, which accumulates in extremely high amounts. This splicing-dependent reversal in CREM function represents an important example of developmental modulation in gene expression.
MeSH Terms
- Aging
- Amino Acid Sequence
- Androgen-Insensitivity Syndrome
- Animals
- Base Sequence
- Brain
- Cyclic AMP Response Element Modulator
- DNA
- DNA-Binding Proteins
- Male
- Mice
- Mice, Mutant Strains
- Molecular Sequence Data
- Oligodeoxyribonucleotides
- Organ Specificity
- Poly A
- Polymerase Chain Reaction
- RNA
- RNA, Messenger
- Repressor Proteins
- Sequence Homology, Nucleic Acid
- Sexual Maturation
- Spermatogenesis
- Testis