Differences in the expression of neurokinin receptor in neural and bone marrow mesenchymal cells: implications for neuronal expansion from bone marrow cells.

The neurokinin-1 (NK-1) receptor interacts with peptides that belong to the tachykinin family. NK-1 is inducible in bone marrow (BM) stroma. In neural cells, its expression is high to constitutive. Screening of three cDNA libraries indicated that this different in NK-1 expression in neural and BM cells could not be explained by differences in the cDNA sequence. Analyses the 5' flanking sequence in BM stroma and three neural cell lines indicated that sequence +1/+358 relative to the transcription start (TS) site could account for the differences in NK-1 expression. Particular cytokines could reverse the repressive effects of region +1/+358 in BM stroma. The effects of NF-kappa B and cAMP activators were studied in stromal cells using a dominant negative inhibitor of NF-kappa B (I kappa B) or a repressor of CRE activators (ICERII gamma). The results showed that their effects of these transcription factors depended on the stimulating cytokine. This study provides insight into the tissue-specific differences in the expression of the NK-1 gene.

The expression of inducible cAMP early repressor (ICER) is altered in prostate cancer cells and reverses the transformed phenotype of the LNCaP prostate tumor cell line.

Inducible cAMP early repressor (ICER) has been shown to be an important mediator of cAMP antiproliferative activity. In this report, it was found that cAMP retards LNCaP cell growth; in contrast, cAMP inhibits the growth of PC-3 and DU-145 cells. ICER protein levels were markedly reduced in prostate cancer epithelial cells and undetectable and uninducible by cAMP in LNCaP and DU 145 cells. Forced expression of ICER in LNCaP cells caused inhibition of cell growth and thymidine incorporation and halted cells at the G(1) phase of the cell cycle. These ICER-bearing LNCaP cells were rendered unable to grow in soft agar and unable to form tumors in nude mice. These results suggest that deregulation of ICER expression may be related to carcinogenesis of the prostate gland.

Mitogen-activated protein kinase phosphorylates and targets inducible cAMP early repressor to ubiquitin-mediated destruction.

Inducible cAMP early repressor (ICER) is an important mediator of cAMP antiproliferative activity that acts as a putative tumor suppressor gene product. In this study, we examined the regulation of ICER protein by phosphorylation and ubiquitination in human choriocarcinoma JEG-3 and mouse pituitary AtT20 cells. We found that cAMP stabilized ICER protein by inhibiting the mitogen-activated protein kinase (MAPK) cascade. Activation of the MAPK pathway increased ICER phosphorylation. ICER phosphorylation was abrogated by inhibition of the MAPK pathway either by cAMP or directly by the MAPK inhibitor PD098059. The MAPKs extracellular signal-regulated kinases 1 and 2 physically interact with ICER and mediated the phosphorylation of ICER on a critical serine residue (Ser-41). A mutant form of ICER in which Ser-41 was substituted by alanine had a half-life 4-5 h longer than its wild-type counterpart. This alteration in stability was due to the inability of the Ser-41-mutant ICER to be efficiently ubiquitinated and degraded via the ubiquitin-proteasome pathway. These results present a novel cell signaling cross-talk mechanism at the cell nucleus between the MAPK and cAMP pathways, whereby MAPK targets a repressor of the cAMP-dependent gene expression for ubiquitination and proteasomal degradation.

Cloning of human preprotachykinin-I promoter and the role of cyclic adenosine 5'-monophosphate response elements in its expression by IL-1 and stem cell factor.

Preprotachykinin-I gene (PPT-I) encodes several peptides with organ-specific functions that link the neuroendocrine-immune-hemopoietic axis. We cloned upstream of the initiation site of human PPT-I promoter and identified consensus sequences for two cAMP response elements (CRE). PPT-I is induced by cytokines including those that signal through the cAMP pathway. Therefore, we studied the role of the two CRE in IL-1alpha and stem cell factor (SCF) stimulation of bone marrow stroma because both cytokines induce endogenous PPT-I in these cells and activate the cAMP pathway. Furthermore, bone marrow stroma expresses the transcription factors regulated by the cAMP pathways such as the repressor (ICERIIgamma) and activator (CREMtau). Mutagenesis of the two CRE and/or cotransfection with vectors that express ICERIIgamma or CREMtau indicated that the two CRE have major roles in PPT-I expression. The two CRE are also required for optimal promoter activity by SCF and IL-1alpha. A particular cytokine could concomitantly induce PPT-I and the high affinity G protein-coupled receptor for PPT-I peptides, NK-1R. We showed that SCF, a representative cytokine, induced PPT-I and NK-1R leading to autocrine and/or paracrine cell activation. Because NK-1R activates cAMP through the G protein, the results suggest that the presence of CRE sequences within PPT-I promoter could be important in the regulation of PPT-I expression by cytokines, irrespective of their ability to signal through cAMP. As PPT-I is implicated in hemopoietic regulation, immune responses, breast cancer, and other neural functions, these studies add to the basic biology of these processes and could provide targets for drug development.

ICER-IIgamma is a tumor suppressor that mediates the antiproliferative activity of cAMP.

The second messenger cAMP inhibits the proliferation of most cell types. The nuclear response of cAMP is mediated by transcription factors like the cAMP-Responsive Element Modulator (CREM) gene. One of the products of the CREM gene, the transcriptional repressor Inducible cAMP Early Repressor-IIgamma (ICER-IIgamma), is induced by cAMP. ICER-IIgamma blocks cells at the G2/M boundary of the cell cycle. Here we show that ICER-IIgamma dramatically inhibits the growth and DNA synthesis of mouse pituitary tumor cells and human choriocarcinoma cells. This alteration in cell growth is coupled with reduced ability of these cells to grow in an anchorage-independent manner and to form tumors in mice. These data demonstrate that ICER-IIgamma is a tumor suppressor gene product mediating the antiproliferative activity of cAMP.

Differential expression of heat shock 70 proteins in primary cultures from rat cerebellum.

While a number of studies have described the heat shock response in established cell lines and in primary cultures of cells derived from the nervous system, there has been no systematic analysis comparing expression and localization of the inducible heat shock 70 (hsp70) proteins and the constitutively synthesized members of the family (hsc70) in neurons and glia. In the present communication, we utilized specific probes to compare the expression of hsp70 and hsc70 mRNAs and proteins in two types of primary cultures, astroglial and neuro-astroglial, from postnatal rat cerebellum. Conditions were adjusted to maintain physiological numbers of microglia in both types of culture, and cultures were analyzed at a number of different time points following a precisely defined heat shock. The northern, in situ hybridization and immunohistochemical analyses resulted in a number of novel observations concerning the nature of the heat shock response in these neuronal and glial cells. In postnatal day 4-5 cultures, hsp70 mRNA levels were elevated for at least 10 h in both types of culture, but in situ hybridization analysis showed no evidence for hsp70 mRNAs in neurons. Microglia were the only cell type in which hsp70 was detected in non-stressed cultures and this cell type contained the highest concentrations of hsp70 proteins in stressed cultures. Hsc70 mRNA levels were also increased after heat shock, but the increase was more transient. Hsc70 mRNAs and proteins were present in all cell types, again with the highest concentrations being present in microglia. Hsc70 mRNAs and proteins were localized in the cytoplasm at all time points examined, with hsc70 protein also being localized in nucleoli. Hsp70 mRNAs and proteins were diffusely localized over nuclei of astrocytes, as well as of most microglia. Hsp70, but not hsc70, was localized on chromosomes in glia once they had resumed cell division after heat shock, suggesting a role for hsp70 either in targeting damaged chromosomal proteins or in cell division. Some cytoplasmic hsp70 was observed in astrocytes of the mixed neuro-astroglial cultures and a delayed hsp70 immunoreactivity was observed in granule neurons in these cultures, suggesting either that translation of low levels of hsp70 mRNAs was more efficient in neurons, or that glial-neuronal translocation of hsp70 proteins had taken place. These results suggest that metabolism and functions of different heat shock protein family members may not always be identical and that care must be taken in extrapolation of results from one cell type to another.

Absence of integrin alpha 6 leads to epidermolysis bullosa and neonatal death in mice.

Cell-extracellular matrix interactions have important roles in many biological processes, including embryonic development, growth control and differentiation. Integrins are the principal receptors for extracellular matrix. They are composed of non-covalently associated alpha and beta chains. Integrin alpha 6 can associate with either beta 1 or beta 4 (refs 2,3). Both integrin complexes are receptors for laminins, major components of basement membranes. The distribution of alpha 6 (refs 4-10) as well as studies using function-blocking antibodies have suggested an essential role for this laminin receptor during embryogenesis, in processes such as endoderm migration or kidney tubule formation9. Here we report that, surprisingly, mice lacking the alpha 6 integrin chain develop to birth. However, they die at birth with severe blistering of the skin and other epithelia, a phenotype reminiscent of the human disorder epidermolysis bullosa. Hemidesmosomes are absent in mutant tissue. This absence is likely to result from the lack of alpha 6/beta 4, the only integrin in hemidesmosomes of stratified squamous and transitional epithelia. Mutations in the genes encoding integrin beta 4 and chains of laminin-5 have been implicated in junctional epidermolysis bullosa. Our study provides evidence that some forms of epidermolysis bullosa may originate from defects of the alpha 6 gene.