Anti-apoptotic gene Bcl2 is required for stapes development and hearing.

In this paper we describe novel and specific roles for the apoptotic regulators Bcl2 and Bim in hearing and stapes development. Bcl2 is anti-apoptotic while Bim is pro-apoptotic. Characterization of the auditory systems of mice deficient for these molecules revealed that Bcl2⁻/⁻ mice suffered severe hearing loss. This was conductive in nature and did not affect sensory cells of the inner ear, with cochlear hair cells and neurons present and functional. Bcl2⁻/⁻ mice were found to have a malformed, often monocrural, porous stapes (the small stirrup-shaped bone of the middle ear), but a normally shaped malleus and incus. The deformed stapes was discontinuous with the incus and sometimes fused to the temporal bones. The defect was completely rescued in Bcl2⁻/⁻Bim⁻/⁻ mice and partially rescued in Bcl2⁻/⁻Bim⁺/⁻ mice, which displayed high-frequency hearing loss and thickening of the stapes anterior crus. The Bcl2⁻/⁻ defect arose in utero before or during the cartilage stage of stapes development. These results implicate Bcl2 and Bim in regulating survival of second pharyngeal arch or neural crest cells that give rise to the stapes during embryonic development.

Identification and characterization of the STIM (stromal interaction molecule) gene family: coding for a novel class of transmembrane proteins.

STIM1 (where STIM is stromal interaction molecule) is a candidate tumour suppressor gene that maps to human chromosome 11p15.5, a region implicated in a variety of cancers, particularly embryonal rhabdomyosarcoma. STIM1 codes for a transmembrane phosphoprotein whose structure is unrelated to that of any other known proteins. The precise pathway by which STIM1 regulates cell growth is not known. In the present study we screened gene databases for STIM1-related sequences, and have identified and characterized cDNA sequences representing a single gene in humans and other vertebrates, which we have called STIM2. We identified a single STIM homologue in Drosophila melanogaster (D-Stim) and Caenorhabditis elegans, but no homologues in yeast. STIM1, STIM2 and D-Stim have a conserved genomic organization, indicating that the vertebrate family of two STIM genes most probably arose from a single ancestral gene. The three STIM proteins each contain a single SAM (sterile alpha-motif) domain and an unpaired EF hand within the highly conserved extracellular region, and have coiled-coil domains that are conserved in structure and position within the cytoplasmic region. However, the STIM proteins diverge significantly within the C-terminal half of the cytoplasmic domain. Differential levels of phosphorylation appear to account for two molecular mass isoforms (105 and 115 kDa) of STIM2. We demonstrate by mutation analysis and protein sequencing that human STIM2 initiates translation exclusively from a non-AUG start site in vivo. STIM2 is expressed ubiquitously in cell lines, and co-precipitates with STIM1 from cell lysates. This association into oligomers in vivo indicates a possible functional interaction between STIM1 and STIM2. The structural similarities between STIM1, STIM2 and D-STIM suggest conserved biological functions.

STIM1: a novel phosphoprotein located at the cell surface.

STIM1 is a novel candidate growth suppressor gene mapping to the human chromosome region 11p15.5 that is associated with several malignancies. STIM1 overexpression studies in G401 rhabdoid tumour, rhabdomyosarcoma and rodent myoblast cell lines causes growth arrest, consistent with a potential role as a tumour growth suppressor. We used highly specific antibodies to show by immunofluorescence and cell surface biotinylation studies that STIM1 is located at the cell surface of K562 cells. Western blot analysis revealed that the 90-kDa STIM1 protein is ubiquitously expressed in various human primary cells and tumour cell lines. STIM1 is not secreted from cells and does not appear to undergo proteolytic processing. We show evidence of post-translational modification of STIM1, namely phosphorylation and N-linked glycosylation. Phosphorylation of STIM1 in vivo occurs predominantly on serine residues. Thus, STIM1, the putative tumour growth suppressor gene is ubiquitously expressed and has features of a regulatory cell-surface phosphoprotein.

Dual posttranscriptional targets of retinoic acid-induced gene expression.

Retinoic acid-induced differentiation of the pre-osteoblastic cell line, UMR 201, is associated with a marked increase in the proficiency of posttranscriptional nuclear processing of alkaline phosphatase mRNA. In this study we attempted to correlate the posttranscriptional actions of retinoic acid with changes in phosphorylation, or abundance of spliceosome components, or both. Treatment with retinoic acid for periods of < or = 4 h resulted in dephosphorylation of nuclear U1 70K protein without affecting its abundance. Peptide mapping showed that U1 70K dephosphorylation was related to the disappearance of one specific phosphopeptide out of four major U1 70K phosphopeptides. A twofold decrease in mRNA expression of an isoform of alternative splicing factor that inhibits splicing was also observed over the same period. Tumor necrosis factor-alpha, which enhances the posttranscriptional action of retinoic acid, reduced U1 70K mRNA expression, while an inhibition of retinoic acid action by transforming growth factor-beta was associated with a marked increase in U1 70K mRNA levels. Our results draw attention to the complex interactions between short- and long-term alterations in the abundance and functional status of U1 70K, as well as SR proteins by growth and/or differentiation factors in the regulation of spliceosome formation and function.

Transcriptional and posttranscriptional regulation of osteopontin gene expression in preosteoblasts by retinoic acid.

This study examines the relative importance of transcriptional and posttranscriptional actions of retinoic acid (RA) in the regulation of osteopontin gene expression in a rat clonal preosteoblastic cell line, UMR 201. Nuclear run-on analysis demonstrated constitutive expression of the osteopontin gene which was increased by threefold after 4 hr treatment with 1 microM RA, returning to a basal level by 24 hr. However, Northern blot analysis, performed concurrently, showed that RA progressively increased the steady-state osteopontin mRNA level beginning 2 hr before any increase in gene transcription and peaking at 24 hr. There was no difference in osteopontin mRNA stability between control and RA-treated cells after gene transcription was inhibited with 5,6-dichloro-1-D-ribofuranosylbenzimidazole (DRB). Total RNA was obtained from cellular subfractions (nuclear matrix, nonmatrix chromatin, nuclear membrane, and cytoplasm) and reverse transcription-polymerase chain reaction (RT-PCR) performed with primers complementary to exons 3 and 4 of the mouse osteopontin gene. Unspliced PCR product, comprising the two exons and the intervening intron, was present in the nuclear matrix fractions of control and RA-treated cells. However, RA resulted in a time-dependent accumulation of mature osteopontin mRNA in all cellular subfractions, suggesting that the proficiency of nuclear processing of primary mRNA transcripts was greatly enhanced by RA. This action depended on de novo protein synthesis. These results demonstrate that the posttranscriptional action of RA is not unique to the regulation of alkaline phosphatase gene expression.

Tumor necrosis factor alpha facilitates nuclear actions of retinoic acid to regulate expression of the alkaline phosphatase gene in preosteoblasts.

This study examines the molecular mechanisms of interaction between tumor necrosis factor alpha (TNF alpha) and retinoic acid on the expression of the alkaline phosphatase gene by rat clonal preosteoblastic cells. In this cell line, alkaline phosphatase mRNA was not constitutively expressed but was progressively induced by treatment with 1 microM retinoic acid, detectable by 6 h. Combining retinoic acid with 0.6 nM TNF alpha resulted in alkaline phosphatase mRNA appearing by 2 h, as well as enhanced expression above that observed with retinoic acid alone at 6, 12, and 24 h. Nuclear run-on analysis showed constitutive transcription of the alkaline phosphatase gene in control and TNF alpha-treated cells. At 4 h, retinoic acid, alone or combined with TNF alpha, increased alkaline phosphatase gene transcriptional rate by 2-fold. However, at 24 h, while no retinoic acid effect was retained, retinoic acid plus TNF alpha resulted in a 5-fold increase in alkaline phosphatase transcriptional rate. Examination of the distribution of nuclear alkaline phosphatase mRNA demonstrated that pre-spliced precursor mRNA was localized to the nuclear matrix in control and all treatment groups. Retinoic acid caused a time-dependent accumulation of mature, spliced alkaline phosphatase mRNA located in the non-matrix and cytoplasmic fractions, implying a post-transcriptional action of retinoic acid in nuclear processing and nucleocytoplasmic transport. Adding TNF alpha with retinoic acid greatly enhanced this effect, which was observed after 4 h, prior to any detectable interaction between TNF alpha and retinoic acid on gene transcription. In sharp contrast, only a negligible amount of nuclear processing occurred in control and TNF alpha-treated cells. This study reveals distinct interactions between TNF alpha and retinoic acid at post-transcriptional as well as transcriptional levels to regulate expression of the alkaline phosphatase gene in preosteoblasts.

Novel action of retinoic acid. Stabilization of newly synthesized alkaline phosphatase transcripts.

Several observations led us to investigate the possibility that retinoic acid achieved its marked induction of alkaline phosphatase gene expression through a post-transcriptional effect in the nuclei of clonal rat pre-osteoblastic UMR 201 cells. The steady-state level of alkaline phosphatase mRNA was significantly stimulated by retinoic acid. Although nuclear run-on analysis showed that 10(-6) M retinoic acid caused an increase in alkaline phosphatase gene transcription, this was transient compared with the rise in alkaline phosphatase mRNA which continued to accumulate for many hours after retinoic acid stimulation of gene transcription had ceased. Moreover, the modest increase in transcriptional rate (approximately 2-fold) was not sufficient to account for the magnitude of the rise in mRNA level. In order, therefore, to examine the influence of retinoic acid on nuclear processing events, a cellular subfractionation method was applied. By nuclease protection analysis, and also by using reverse transcription-polymerase chain reaction, sequences corresponding to intron 2 and intron 4, respectively, were demonstrated specifically in the nuclear matrix fraction of both control and retinoic acid-treated cells. Mature (spliced) alkaline phosphatase mRNA accumulated in the non-matrix (DNase I/salt eluate, nuclear membrane) and cytoplasmic fractions of retinoic acid-treated cells at more than 100-fold greater levels than in control cells. This implies that nuclear processing of the primary RNA transcript occurred only in cells treated with retinoic acid. The post-transcriptional action of retinoic acid was inhibited by cotreatment with 10 micrograms/ml cycloheximide. Transforming growth factor beta (TGF beta) (1 ng/ml) did not influence whole cell alkaline phosphatase levels in UMR 201 cells. Nevertheless, TGF beta increased the transcriptional rate of the alkaline phosphatase gene. Although precursor mRNA was detected in the nuclear matrix fraction of TGF beta-treated cells, there was no evidence of further mRNA nuclear processing. The data are consistent with stabilization of nascent alkaline phosphatase mRNA chains by retinoic acid treatment and suggests that regulation of mRNA processing can be independent of gene transcription. This study demonstrates a novel post-transcriptional action of retinoic acid which plays an important, if not a dominant role, in determining the steady-state level of alkaline phosphatase mRNA.

Opposing influences of glucocorticoid and retinoic acid on transcriptional control in preosteoblasts.

UMR 201 is a nontransformed rat clonal cell line derived from neonatal calvaria with phenotypic characteristics of preosteoblasts. Retinoic acid strongly induces expression of alkaline phosphatase and its mRNA in these cells. Dexamethasone substantially reduced the retinoic acid-induced expression of alkaline phosphatase. This apparent interaction between dexamethasone and retinoic acid effects raised the possibility that interactions may extend to other osteoblast-related phenotypic characteristics in UMR 201 cells. Treatment with dexamethasone resulted in a decrease in the expression of mRNA for pro-alpha 1(I) collagen, but upon coincubation with 1 microM retinoic acid for 24 h, the decrease in mRNA for pro-alpha 1(I) collagen was abrogated. Dexamethasone (Dex) treatment caused a dose-dependent increase in osteonectin mRNA, half maximally effective between 1 nM and 10 nM Dex. One micromolar of retinoic acid alone led to a small increase in expression of osteonectin mRNA but prevented any further increase when Dex was added to retinoic acid-treated cells. To study transcriptional control, osteonectin genomic fragments were linked to the bacterial reporter gene, chloramphenicol acetyltransferase, and introduced by transfection into UMR 201 cells. Dexamethasone increased the transcriptional activity of an osteonectin-chloramphenicol acetyltransferase construct; 100 nM Dex resulted in a 3-fold increase over control cells which was attenuated when 1 microM retinoic acid was added to the incubation, while retinoic acid alone resulted in a 2-fold increase in transcriptional activity. Finally, it was noted that coincubation with retinoic acid and Dex stimulated the proliferation of UMR 201 cells when compared with either treatment alone. This study shows the potential importance of hormonal interactions in the expression of osteoblast function.

Retinoic acid and tumour necrosis factor-alpha act in concert to control the level of alkaline phosphatase mRNA.

Retinoic acid has a specific role in cellular differentiation and is believed to act by regulating the transcription of specific genes. In the present work, evidence is provided to show that alkaline phosphatase (ALP) gene expression is mediated by retinoic acid in a model clonal cell line (UMR 201) derived from rat neonatal calvaria. These cells have the characteristics of relatively undifferentiated mesenchymal cells with a very low basal ALP activity which is dramatically increased by retinoic acid. Messenger RNA for ALP was clearly demonstrated when the cells were treated with 1 microM retinoic acid for 24 h. Recombinant human tumour necrosis factor-alpha (recombinant TNF-alpha) interacted with retinoic acid to potentiate the rise in ALP activity, although recombinant TNF-alpha alone had no effect. The potentiation of retinoic acid-induced ALP activity was correlated with an increased amount of mRNA for ALP with the combined treatment. By observing the rate of decay of mRNA for actin and ALP, we were able to demonstrate that the interaction between retinoic acid and recombinant TNF-alpha modulated the steady state of ALP mRNA. The mode of action of recombinant TNF-alpha may serve as a model for other paracrine regulators of cell function.