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Phorbol esters and cAMP differentially regulate the expression of CD4 and CD8 in human thymocytes.

Martinez-Valdez H, Madrid-Marina V, Cohen A - BMC Immunol. (2002)

Bottom Line: Determining the intracellular signals that differentially regulate the expression of CD4 and CD8 is important to understand the mechanisms that are implicated in selection of single positive CD4+CD8- or CD4-CD8+.The present study shows that stimulation of human thymocytes by phorbol esters or cAMP result in a differential regulation of CD4 and CD8 expression, both at the mRNA and cell surface glycoprotein level.The differential regulation of CD4 and CD8 gene expression suggests that the selective activation of protein kinase C (PKC) and cAMP-dependent protein kinases (PKA) may be required for the selection of single positive CD4+CD8- and CD4-CD8+ cells during Intrathymic differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology, Box 178, The University of Texas MD Anderson Cancer Center, USA. hmartine@mdanderson.org

ABSTRACT

Background: Intrathymic development and selection of the T lymphocyte repertoire is restricted by the interactions of the T cell antigen receptor and CD4 or CD8 co-receptors with self major histocompatibility complex molecules. Positive or negative selection depends on a tight regulatory control of CD4 and CD8 expression. Determining the intracellular signals that differentially regulate the expression of CD4 and CD8 is important to understand the mechanisms that are implicated in selection of single positive CD4+CD8- or CD4-CD8+.

Results: The present study shows that stimulation of human thymocytes by phorbol esters or cAMP result in a differential regulation of CD4 and CD8 expression, both at the mRNA and cell surface glycoprotein level.

Conclusions: The differential regulation of CD4 and CD8 gene expression suggests that the selective activation of protein kinase C (PKC) and cAMP-dependent protein kinases (PKA) may be required for the selection of single positive CD4+CD8- and CD4-CD8+ cells during Intrathymic differentiation.

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Northern blot to examine the effect of cyclic AMP analogs and cholera toxin on CD4, CD8, and CD3 mRNA levels in human thymocytes. Experimental conditions are number-coded and respectively identified
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Figure 3: Northern blot to examine the effect of cyclic AMP analogs and cholera toxin on CD4, CD8, and CD3 mRNA levels in human thymocytes. Experimental conditions are number-coded and respectively identified

Mentions: On the other hand, it has been previously demonstrated that direct activation of adenylate cyclase by Forskolin or cholera toxin, inhibition of cAMP-specific phosphodiesterase, and treatment with a cell permeable cAMP analog, induces a rise in intracellular cAMP [22-26]. Since cAMP is known to modulate the expression of CD8 in mice [27], we sought to determine whether cAMP-dependent protein kinase (PKA)-mediated signaling plays a role in modulating the mRNA levels of CD3, CD4, and CD8 in ex vivo examined human thymocytes. Fig. 3 shows that the signal, which regulates the expression of human CD8, is cAMP-mediated, since direct activation of adenylate cyclase by cholera toxin produced the same effects as the di-butiryl cAMP analog. The requirement of phosphodiesterase inhibition by IBMX further supports this statement. As it can also be observed, cAMP coordinately regulated the expression of both the α and β chains of the CD8 molecule. CD4 remained unchanged and CD3 was minimally affected, thus stressing the selectivity of the cAMP-dependent down-regulation of CD8 expression (Fig. 3). To further investigate whether PMA and cAMP regulate CD4 and CD8 expression at the protein level, surface detection of the CD3, CD4, and CD8 was also assessed. As observed in Fig. 4a PMA dropped the proportion of CD4+ cells from 60% to zero, produced a slight decrease (from 71% to 64%) in CD8+ cells, and did not affect the expression of CD3. cAMP on the other hand had little effect on the surface expression of the glycoproteins. These findings are in contrast with the drastic down-regulation of CD8 transcript levels exerted by cAMP. However, regulation gene expression is complex and engages numerous checkpoints that include transcription, RNA processing, nuclear/cytosol transport, translation, and RNA and Protein stability. Since the half-lives of mRNA and protein can be dramatically different, we hypothesize that an increased CD8 protein stability may result in a delay of CD8 surface disappearance, thus favoring its transient selection. As CD8 gene transcription has been turned-off by cAMP-mediated signaling, surface CD8 subsequently fades with the concomitant emergence of CD4+ cells. In keeping with this rationale, the kinetics of the PMA-mediated down-regulation of CD4 (Fig. 4b) reveals a sharp and rapid decrease on CD4 surface expression as early as 15 min. of incubation with PMA, with a complete disappearance by 2.0 hr. The relatively short kinetics of surface CD4 down-regulation and the delayed CD8 surface disappearance can be interpreted as a PKC-mediated selection signaling in favor of CD8+CD4-/α:β-TCR cells that may operate during intrathymic T cell differentiation. Binding of specific ligands to CD4 (including phorbol esters) results in internalization of the molecule by endocytosis, which in turn is phosphorylated [28] and possibly immobilized. It is conceivable that a similar mechanism might be used by extra-cellular signals in the thymic epithelium during intrathymic differentiation.


Phorbol esters and cAMP differentially regulate the expression of CD4 and CD8 in human thymocytes.

Martinez-Valdez H, Madrid-Marina V, Cohen A - BMC Immunol. (2002)

Northern blot to examine the effect of cyclic AMP analogs and cholera toxin on CD4, CD8, and CD3 mRNA levels in human thymocytes. Experimental conditions are number-coded and respectively identified
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC65519&req=5

Figure 3: Northern blot to examine the effect of cyclic AMP analogs and cholera toxin on CD4, CD8, and CD3 mRNA levels in human thymocytes. Experimental conditions are number-coded and respectively identified
Mentions: On the other hand, it has been previously demonstrated that direct activation of adenylate cyclase by Forskolin or cholera toxin, inhibition of cAMP-specific phosphodiesterase, and treatment with a cell permeable cAMP analog, induces a rise in intracellular cAMP [22-26]. Since cAMP is known to modulate the expression of CD8 in mice [27], we sought to determine whether cAMP-dependent protein kinase (PKA)-mediated signaling plays a role in modulating the mRNA levels of CD3, CD4, and CD8 in ex vivo examined human thymocytes. Fig. 3 shows that the signal, which regulates the expression of human CD8, is cAMP-mediated, since direct activation of adenylate cyclase by cholera toxin produced the same effects as the di-butiryl cAMP analog. The requirement of phosphodiesterase inhibition by IBMX further supports this statement. As it can also be observed, cAMP coordinately regulated the expression of both the α and β chains of the CD8 molecule. CD4 remained unchanged and CD3 was minimally affected, thus stressing the selectivity of the cAMP-dependent down-regulation of CD8 expression (Fig. 3). To further investigate whether PMA and cAMP regulate CD4 and CD8 expression at the protein level, surface detection of the CD3, CD4, and CD8 was also assessed. As observed in Fig. 4a PMA dropped the proportion of CD4+ cells from 60% to zero, produced a slight decrease (from 71% to 64%) in CD8+ cells, and did not affect the expression of CD3. cAMP on the other hand had little effect on the surface expression of the glycoproteins. These findings are in contrast with the drastic down-regulation of CD8 transcript levels exerted by cAMP. However, regulation gene expression is complex and engages numerous checkpoints that include transcription, RNA processing, nuclear/cytosol transport, translation, and RNA and Protein stability. Since the half-lives of mRNA and protein can be dramatically different, we hypothesize that an increased CD8 protein stability may result in a delay of CD8 surface disappearance, thus favoring its transient selection. As CD8 gene transcription has been turned-off by cAMP-mediated signaling, surface CD8 subsequently fades with the concomitant emergence of CD4+ cells. In keeping with this rationale, the kinetics of the PMA-mediated down-regulation of CD4 (Fig. 4b) reveals a sharp and rapid decrease on CD4 surface expression as early as 15 min. of incubation with PMA, with a complete disappearance by 2.0 hr. The relatively short kinetics of surface CD4 down-regulation and the delayed CD8 surface disappearance can be interpreted as a PKC-mediated selection signaling in favor of CD8+CD4-/α:β-TCR cells that may operate during intrathymic T cell differentiation. Binding of specific ligands to CD4 (including phorbol esters) results in internalization of the molecule by endocytosis, which in turn is phosphorylated [28] and possibly immobilized. It is conceivable that a similar mechanism might be used by extra-cellular signals in the thymic epithelium during intrathymic differentiation.

Bottom Line: Determining the intracellular signals that differentially regulate the expression of CD4 and CD8 is important to understand the mechanisms that are implicated in selection of single positive CD4+CD8- or CD4-CD8+.The present study shows that stimulation of human thymocytes by phorbol esters or cAMP result in a differential regulation of CD4 and CD8 expression, both at the mRNA and cell surface glycoprotein level.The differential regulation of CD4 and CD8 gene expression suggests that the selective activation of protein kinase C (PKC) and cAMP-dependent protein kinases (PKA) may be required for the selection of single positive CD4+CD8- and CD4-CD8+ cells during Intrathymic differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Immunology, Box 178, The University of Texas MD Anderson Cancer Center, USA. hmartine@mdanderson.org

ABSTRACT

Background: Intrathymic development and selection of the T lymphocyte repertoire is restricted by the interactions of the T cell antigen receptor and CD4 or CD8 co-receptors with self major histocompatibility complex molecules. Positive or negative selection depends on a tight regulatory control of CD4 and CD8 expression. Determining the intracellular signals that differentially regulate the expression of CD4 and CD8 is important to understand the mechanisms that are implicated in selection of single positive CD4+CD8- or CD4-CD8+.

Results: The present study shows that stimulation of human thymocytes by phorbol esters or cAMP result in a differential regulation of CD4 and CD8 expression, both at the mRNA and cell surface glycoprotein level.

Conclusions: The differential regulation of CD4 and CD8 gene expression suggests that the selective activation of protein kinase C (PKC) and cAMP-dependent protein kinases (PKA) may be required for the selection of single positive CD4+CD8- and CD4-CD8+ cells during Intrathymic differentiation.

Show MeSH
Related in: MedlinePlus