Jose Miguel Cosgaya

Nerve growth factor and ras regulate β-amyloid precursor protein gene expression in PC12 cells

Abstract: The β‐amyloid protein, the major component of the vascular and plaque amyloid deposits that characterize Alzheimers disease, derives from a larger β‐amyloid precursor protein (APP) that is expressed in both neural and nonneural cells. An increased expression of APP might actively contribute to the development of the pathology; however, the mechanisms involved in the regulation of APP gene expression are not yet well understood. In PC12 cells, a rat pheochromocytoma cell line, we have demonstrated that nerve growth factor (NGF) induces the APP gene expression and increases APP mRNA levels in the presence of 0.5 or 15% serum. Expression of activated ras in the PC12 cell subline UR61 also leads to a significant increase in content of APP transcripts, and a dominant negative mutant of ras blocks the NGF‐induced response. Other ligands of tyrosine kinase receptors, such as fibroblast growth factor, which causes morphological differentiation, or epidermal growth factor, which induces cell growth, also increase APP mRNA levels in PC12 cells. These results suggest that ras mediates the induction of APP gene expression by NGF and other ligands of tyrosine kinase receptors.

Comparison of the effects of retinoic acid and nerve growth factor on PC12 cell proliferation, differentiation, and gene expression.

Abstract: Retinoic acid (RA) produced a dose‐dependent inhibition of PC12 cell growth and the appearance of cell clusters without neurite extension. RA‐induced cell clumping was similar to that caused by dexamethasone (Dx). Nerve growth factor (NGF) induced neurite extension, and the combination of RA plus NGF produced a maximal decrease in cell proliferation with a mixed morphology in which part of the cell population had neurites and part formed clumps. Transcriptional effects of RA were demonstrated by the increase in the activity of reporter constructs that contain an RA response element. RA also regulated expression of endogenous genes in PC12 cells. The retinoid produced a two‐ to threefold increase in level of p75LNGFR mRNA (the low‐affinity NGF receptor), without altering expression of the trk protooncogene (the high‐affinity NGF receptor carrying tyrosine kinase activity). RA also caused a transient increase in level of tyrosine hydroxylase (TH) mRNA (twofold after 16 h), which returned to basal levels and then decreased relative to basal levels at 48 h. The effect of NGF on the expression of these genes was identical to that produced by RA. However, incubation with Dx did not induce p75LNGFR mRNA and produced a strong and sustained increase of TH mRNA level (three‐ to fivefold after 48 h). These results show that, despite the common morphological changes produced by RA and glucocorticoids in PC12 cells, the biochemical changes caused by RA are similar to those produced by NGF. Therefore, RA could initiate a biochemical program of neuronal differentiation in PC12 cells, although a fully differentiated phenotype with neurite extension is not obtained.

Retinoic acid induces secretion of transforming growth factors by PC12 pheochromocytoma cells

Conditioned medium from PC12 cells incubated with retinoic acid (RA) increases [3H]thymidine incorporation in normal rat kidney (NRK) fibroblasts and 3D9 epithelial cells. The medium also causes anchorage-independent growth of NRK cells, which is strongly potentiated either in the presence of EGF or after activation of latent forms of transforming growth factors (TGFs) by acidification. These results suggest that RA regulates the release of more than one growth factor by PC12 cells. Conditioned media from control or NGF-treated PC12 cells causes growth of NRK cells in soft agar only after acidification. An increase in expression of the TGF-β1 gene is coincident with NGF-induced neuronal differentiation of PC12 cells. In addition, RA also causes a dose- and time-dependent increase in content of TGF-β1 transcripts. This increase is, at least in part, secondary to transcriptional activation. Sequences responsible for the effect of RA and NGF are located in the 5′-flanking region of the TGF-β1 gene. The TFG-β1 gene has two promoters and in transient transfection assays RA and NGF significantly enhance the activity of constructs containing the second promoter. High-affinity TGF-β1 receptors were undetectable in PC12 cells both before and after NGF or RA treatment. RA and NGF decrease PC12 cell proliferation and a neutralizing anti-TGF-β1 antibody does not reverse this inhibition. In summary, an increase in expression and secretion of TGF-β1 accompanies RA and NGF-induced PC12 cell growth arrest, but TGF-β1 does not play an autocrine role in this inhibition.

Nerve Growth Factor Regulates Transforming Growth Factor‐β1 Gene Expression by Both Transcriptional and Posttranscriptional Mechanisms in PC12 Cells

Abstract: PC12 cells express two transforming growth factor (TGF)‐β1 transcripts, 1.7 and 2.5 kb in size. Other TGF‐βs are expressed at much lower levels. Incubation with nerve growth factor (NGF) produced a time‐ and dose‐dependent increase in content of TGF‐β1 transcripts. The level of the smaller mRNA increased little, whereas that of the 2.5‐kb transcript increased more, with the latter becoming the predominant TGF‐β1 message. NGF differentially regulated the stability of both mRNAs. The half‐life of the 2.5‐kb transcript was not altered by NGF; however, the half‐life of the 1.7‐kb mRNA was ∼6 h and increased to >30 h on incubation with NGF. In addition, induction of the 2.5‐kb TGF‐β1 mRNA by NGF required de novo protein synthesis, whereas induction of the 1.7‐kb TGF‐β1 mRNA was independent of protein synthesis. The NGF effect was independent of protein kinase C activation, which also preferentially induced the larger transcript. PC12 cells release a significant amount of TGF‐β1, and incubation with NGF further increases TGF‐β1 production. This factor is released in a latent form. These results indicate that an increase in expression and secretion of TGF‐β1 accompanies neuronal differentiation in PC12 cells. Regulation of TGF‐β1 gene expression by NGF is complex, involving both increased transcription of the TGF‐β1 gene and stabilization of the smaller TGF‐β1 transcript.

Retinoic acid regulates myelin formation in the peripheral nervous system

Understanding the mechanisms that control myelin formation is essential for the development of demyelinating diseases treatments. All‐trans‐retinoic acid (RA) plays an essential role during the development of the nervous system as a potent regulator of morphogenesis, cell growth, and differentiation. In this study, we show that RA is also a potent inhibitor of peripheral nervous system (PNS) myelination. RA acts through its binding to RA receptors (RAR) and retinoid X receptors (RXR), two members of the superfamily of nuclear receptors that act as ligand‐dependent transcription factors. Schwann cells (SCs) express all retinoid receptors during the relevant stages of myelin formation. Through the activation of RXR, RA produces an upregulation of Krox20, a SC‐specific regulatory transcription factor that plays a central role during myelination. Krox20 upregulation translates into Mbp and Mpz overexpression, therefore blocking myelin formation. This increase in myelin protein expression is accompanied by the induction of an adaptive ER stress response. At the same time, through a RAR‐dependent mechanism, RA downregulates myelin‐associated glycoprotein, which also contributes to the dysmyelinating effect of the retinoid.

Synergy between RA and TLR3 promotes type I IFN- dependent apoptosis through upregulation of TRAIL pathway in breast cancer cells

Due to its ability to regulate the growth, differentiation and apoptosis of cancer cells, retinoic acid (RA) is considered a signaling molecule with promising therapeutic potential in oncology. In this study, we show that RA is able to induce the intrinsic ability of breast cancer cells to recognize double-stranded RNA (dsRNA) through the upregulation of Toll-like receptor 3 (TLR3) expression. RA, co-administered with the dsRNA mimicker polyinosinic–polycytidylic acid (poly(I:C)), synergizes to mount a specific response program able to sense dsRNA through the concurrent upregulation of TLR3, the dsRNA helicases melanoma differentiation-associated antigen-5 (MDA-5) and RA-inducible gene-1 (RIG-1), and the dsRNA-activated protein kinase (PKR) expression, leading breast cancer cells to specifically express downstream transcriptional targets of dsRNA sensors, such as interferon-β (IFNβ), interleukin-8 (IL-8), chemokine (C-C motif) ligand 5 (CCL5), and C-X-C motif Chemokine 10 (CXCL10). A TLR3-dependent apoptotic program is also induced by RA and poly(I:C) co-treatment that correlates with the induction of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) and contributes to block breast cancer cell proliferation. The mechanisms of apoptosis induced by RA/poly(I:C) in breast cancer cells involve type I IFN autocrine signaling, caspase-8 and caspase-3 activation, as well as TRAIL signaling. Our results reveal important links among RA, TLR3 and TRAIL and highlight the combined use of RA and poly(I:C) as a potential effective tumor therapy by improving the apoptotic response of cancer cells with low sensitivity to the action of synthetic dsRNA.

Nerve growth factor activates the RARβ2 promoter by a Ras-dependent mechanism

Nerve growth factor (NGF) and retinoic acid (RA) exert important actions on PC12 cells. We have previously shown that incubation with NGF induces retinoic acid receptor β (RARβ) binding to a hormone response element in PC12 cells. In this study we show that NGF increases RARβ protein levels by enhancing basal RARβ2 promoter activity, and potentiates stimulation by RA in transient transfection assays. The effect of RA is mediated by a RA response element (RARE) located at −37/−53 and mutation of this element abolishes activation by the retinoid, as well as cooperation with NGF. However, the action of NGF is independent of the RARE and is mediated by sequences overlapping the TATA box and the INR comprising nucleotides −59 to +14. NGF produces a strong decrease in some of the complexes that bind to the INR. These results suggest that the RARβ2 gene could be in a basal repressed state and NGF could increase RARβ2 transcription by inducing the release of some inhibitory factors from the INR. Functional Ras is required for RARβ2 promoter activation by NGF because expression of oncogenic Ras increases promoter activity and a dominant inhibitory Ras mutant blocks the effect of NGF. Oncogenic Raf also mimics the effect of NGF on the promoter. Other ligands of tyrosine kinase receptors that stimulate Ras also cause RARβ2 promoter activation and act cooperatively with RA. These results indicate the existence of cross‐coupling of the Ras–Raf signal transduction pathway with retinoid receptor pathways which could increase sensitivity to RA and be important for PC12 cell function.

Influence of Ras and retinoic acid on nerve growth factor induction of transin gene expression in PC12 cells

Nerve growth factor (NGF)- and ras-induced neuronal differentiation of PC12 cells is accompanied by expression of transin, a secreted metalloproteinase. Retinoic acid (RA) is known to exert important effects on neural cell proliferation and differentiation. In this study we have analysed different PC12 sublines which express either activated Ras or dominant negative p21N17 Ras, to evaluate the influence of retinoic acid (RA) on the response of the transin gene to NGF and Ras. There was a good correlation between neurite extension and induction of transin mRNA levels in the different subclones. NGF did not induce transin mRNA in cells which do not differentiate in response to this neurotrophin. In addition, incubation with RA did not detectably increase basal transin mRNA levels, but caused a significant increase in the transin response to NGF or Ras in cells in which these factors induce a neuronal morphology. Sequences contained within 750 base pairs of the 5′ flanking region of the transin gene confer responsiveness to NGF and Ras, but do not mediate the stimulatory effect of RA. In addition, expression of oncogenic Raf increases transin promoter activity in PC12 cells, but a dominant-negative Raf mutant was unable to block NGF-induced transin activity suggesting the existence of a bifurcation downstream of ras in the signaling mechanism leading to transin expression by NGF.

Retinoic acid regulates selectively the expression of immediate early response genes in PC12 cells

Nerve growth factor (NGF) induces neuronal differentiation and growth arrest in PC12 cells. One of the initial effects of NGF in these cells is the induction of the expression of immediate early genes (IEGs). In this study we have analyzed the influence of retinoic acid (RA), which exerts important effects on PC12 cell proliferation and function, on the expression of IEGs. Incubation with RA did not alter NGFI‐A mRNA levels, but significantly reduced the NGFI‐B and c‐fos response to NGF and serum. The response to NGF was maximal in the presence of cycloheximide, and RA also reduced the superinduction of NGFI‐B and c‐fos mRNA levels. Sequences contained within the 5′ flanking region of the c‐fos gene confer responsiveness to NGF and mediate the inhibitory effect of RA. The differential regulation by RA suggests that NGF induces expression of the three IEGs by different mechanisms.

Regulation of Retinoid Receptors by Retinoic Acid and Axonal Contact in Schwann Cells

Background Schwann cells (SCs) are the cell type responsible for the formation of the myelin sheath in the peripheral nervous system (PNS). As retinoic acid (RA) and other retinoids have a profound effect as regulators of the myelination program, we sought to investigate how their nuclear receptors levels were regulated in this cell type. Methodology/Principal Findings In the present study, by using Schwann cells primary cultures from neonatal Wistar rat pups, as well as myelinating cocultures of Schwann cells with embryonic rat dorsal root ganglion sensory neurons, we have found that sustained expression of RXR-γ depends on the continuous presence of a labile activator, while axonal contact mimickers produced an increase in RXR-γ mRNA and protein levels, increment that could be prevented by RA. The upregulation by axonal contact mimickers and the transcriptional downregulation by RA were dependent on de novo protein synthesis and did not involve changes in mRNA stability. On the other hand, RAR-β mRNA levels were only slightly modulated by axonal contact mimickers, while RA produced a strong transcriptional upregulation that was independent of de novo protein synthesis without changes in mRNA stability. Conclusions/Significance All together, our results show that retinoid receptors are regulated in a complex manner in Schwann cells, suggesting that they could have a prominent role as regulators of Schwann cell physiology.