Induction of c-fos in pituitary cells by thyrotrophin-releasing hormone and phorbol 12-myristate 13-acetate depends upon Ca2+ influx

Calcium-containing crystals such as basic calcium phosphate (BCP) 1 and calcium pyrophosphate dihydrate (CPPD) are two of the most common forms of pathologic articular materials that are associated with destructive arthropathies involving cartilage degeneration (1, 2). At concentrations found in pathologic human joint fluids, these crystals exert biological effects on cultured cells in a manner similar to growth factors like platelet-derived growth factor, epidermal growth factor, and serum. It has been demonstrated that BCP crystals stimulate fibroblast, synoviocyte, and chondrocyte mitogenesis in vitro (3); stimulate the production of prostaglandin via the phospholipase A 2 /cyclo-oxygenase pathway (4); activate phospholipase C and inositol phospholipid hydrolysis (5); induce the expression of the proto-oncogenes, c-fos and c-myc (6, 7); and induce the synthesis and secretion of metalloproteinases (MMPs) 1, 3, 8, and 13 (8 -12).In contrast to other mitogenic and growth factors, BCP crystal-elicited signal transduction pathways have not been completely studied. However, we have identified some of the component molecules involved in calcium-containing crystal signal transduction mechanisms. One pathway activated upon crystal stimulation of human fibroblasts (HF) is the p44 and p42 mitogen-activated protein kinase (p44/42 MAPK) pathway, also known as extracellular signal-related mitogen protein kinases 1 and 2 (ERK1 and ERK2), respectively. The MAPK cascade can be blocked by the selective inhibitors, PD98059 (13) and U0126 (14), which hinder the activation and phosphorylation of MEK (MAPK/ERK kinase). Co-treatment of HF with BCP crystals and PD98059 blocks crystal-induced p44/42 MAPK activation and mitogenesis (15) in addition to crystalinduced up-regulation of MMP-1 and MMP-3 mRNA and protein expressions (16). Moreover, phosphocitrate (PC), a specific inhibitor of the biological effects of BCP and CPPD crystals (17), also blocks crystal-induced activation of p44/42 MAPK, further supporting the role of this signal pathway in crystalinduced responses in HF (15).Another messenger with an apparent role in crystal-activated signal transduction is calcium. We have previously shown that treatment of HF with BCP crystals induces a rapid transient rise of intracellular calcium levels in seconds due to calcium influx from outside the cell, followed by a slow and sustained increase of intracellular calcium within 60 min after stimulation, due to crystal dissolution (18). Removal of calcium from the cell culture medium attenuates the BCP crystal in-

Section: Discussionsupporting

confidence: 86%

Molecular Mechanism of the Induction of Metalloproteinases 1 and 3 in Human Fibroblasts by Basic Calcium Phosphate Crystals

Reuben

Brogley

Sun

et al. 2002

“…GH4C1 cells stimulated by TRH in medium with low free extracellular Ca 2ϩ result in the suppression of sustained intracellular Ca 2ϩ signals. (32,36). We demonstrated marked differences in MKP-1 gene expression in the absence or presence of extracellular Ca 2ϩ after TRH stimulation, which defines a physiological role for calcium in MKP-1 induction associated with the release of the block to elongation.…”

Section: -41mentioning

confidence: 72%

“…Previous reports have shown that the prolonged phase of [Ca 2ϩ ] i spiking was essential to enhance and maintain proto-oncogene mRNA levels over time (31,32,36 A, the rat MKP-1 gene was amplified by PCR, cloned, and sequenced (GenBank TM accession number AF357203), as described under "Experimental Procedures." The cloned fragment of 2.9 kilobase pairs was used to prepare random labeled DNA probes (probes 1-5) for Southern analysis.…”

Section: Induction Of Mkp-1 Mrna By Trh Depends Upon Ca 2ϩmentioning

confidence: 99%

MAP Kinase Phosphatase-1 Gene Transcription in Rat Neuroendocrine Cells Is Modulated by a Calcium-sensitive Block to Elongation in the First Exon

Ryser¹,

Tórtola²,

Haasteren³

et al. 2001

Transcriptional elongation of many eukaryotic, prokaryotic, and viral genes is tightly controlled, which contributes to gene regulation. Here we describe this phenomenon for the MAP kinase phosphatase 1 (MKP-1) immediate early gene. In rat GH4C1 pituitary cells, MKP-1 mRNA is rapidly and transiently induced by the thyrotropin-releasing hormone (TRH) and the epidermal growth factor EGF via transcriptional activation of the gene. Ca 2؉ signals are necessary for the induction of MKP-1 in response to TRH but not to EGF. Reporter gene analysis with the newly cloned rat promoter sequence shows only limited induction in response to various stimuli, including TRH or EGF. By nuclear run-on assays we demonstrate that in basal conditions, a strong block to elongation in the first exon regulates the MKP-1 gene and that stimulation with either TRH or EGF overcomes the block. Ca 2؉ signals are important to release the MKP-1 elongation block in a manner similar to the c-fos oncogene. These results suggest that a common mechanism of intragenic regulation may be conserved between MKP-1 and c-fos in mammalian cells.Long term cellular processes such as proliferation, differentiation, and neuronal plasticity are controlled by extracellular stimuli and require the synthesis of new gene products. Following stimulation, expression of immediate early genes (IEGs) precedes the expression of late response genes, the latter encoding for proteins implicated in specific functions. The best known IEG products are transcription factors such as c-Fos, c-Jun, and c-Myc, which control the expression of late response genes (1, 2). Not all IEGs encode for transcription factors. For instance, structural proteins like actin or tropomyosin, cytokines, and other regulatory proteins show a rapid and transient induction by growth factors (1).Recently, a group of dual specificity phosphatases have been identified as being IEG products induced by various stimuli (growth factors, stress, neurotransmittors, etc.; reviewed in Ref.3). These dual specificity (threonine/tyrosine) phosphatases have been named MAP kinase phosphatases (DSPs or MKPs), since they are effective in the inactivation of MAP kinases by dual dephosphorylation (4). MAP kinase phosphatase-1 (MKP-1/CL100/3CH134) is one example of this group of nuclear enzymes encoded by an IEG. Although MKP-1 gene transcription is activated by multiple signals, such as mitogens (5, 6), cytokines (7), oxidative stress (8), heat shock (8), or hypoxia (9), the precise mode of gene regulation of this immediate early gene by such stimuli remains unclear. A comparison of the 5Ј-flanking sequence of the murine and the human genes revealed two conserved Ca 2ϩ /cAMP-responsive elements (CREs) and one E box motif in the promoter region of MKP-1. Recently, the upstream stimulatory factor, a member of the basic/helix-loop-helix/leucine zipper family has been shown to bind to the E box motif and transactivate MKP-1 expression in synergy with protein kinase A (10). Since multiple intracellular signals can target MKP-1 gen...

“…Ϫ fibroblasts to agents that elevate intracellular levels of calcium and/or cAMP, since these pathways were shown to synergize in c-fos activation (46,47). In Ltk Ϫ cells, PMA, IBMX ϩ 8-Br-cAMP, or calcium ionophore (A23187) generated only a minor induction of c-fos mRNA (Fig.…”

Section: C-fos Induction In Ltk ϫ Cells Requires the Concerted Actionmentioning

confidence: 98%

A Novel Calcium Signaling Pathway Targets the c-fosIntragenic Transcriptional Pausing Site

Coulon¹,

Veyrune²,

et al. 1999

In many cell types, increased intracellular calcium gives rise to a robust induction of c-fos gene expression. Here we show that in mouse Ltk ؊ fibroblasts, calcium ionophore acts in synergy with either cAMP or PMA to strongly induce the endogenous c-fos gene. Run-on analysis shows that this corresponds to a substantial increase in active polymerases on downstream gene sequences, i.e. relief of an elongation block by calcium. Correspondingly a chimeric gene, in which the human metallothionein promoter is fused to the fos gene, is strongly induced by ionophore alone, unlike a c-fos promoter/␤-globin coding unit chimeric construct. Internal deletions in the hMT-fos reporter localize the intragenic calcium regulatory element to the 5 portion of intron 1, thereby confirming and extending previous in vitro mapping data. Ionophore induced cAMP response element-binding protein phosphorylation on Ser 133 without affecting the extracellular signal-regulated kinase cascade. Surprisingly, induction involved neither CaM-Ks nor calcineurin, while the calmodulin antagonist W7 activated c-fos transcription on its own. These data suggest that a novel calcium signaling pathway mediates intragenic regulation of c-fos expression via suppression of a transcriptional pause site.