Multiple phenotypes in adult mice following inactivation of the Coxsackievirus and Adenovirus Receptor (Car) gene.

To determine the normal function of the Coxsackievirus and Adenovirus Receptor (CAR), a protein found in tight junctions and other intercellular complexes, we constructed a mouse line in which the CAR gene could be disrupted at any chosen time point in a broad spectrum of cell types and tissues. All knockouts examined displayed a dilated intestinal tract and atrophy of the exocrine pancreas with appearance of tubular complexes characteristic of acinar-to-ductal metaplasia. The mice also exhibited a complete atrio-ventricular block and abnormal thymopoiesis. These results demonstrate that CAR exerts important functions in the physiology of several organs in vivo.

Conditional expression of a glucocorticoid receptor transgene in thymocytes reveals a role for thymic-derived glucocorticoids in thymopoiesis in vivo.

We and others have previously reported that thymic epithelial cells produce glucocorticoids (GCs). In vitro studies have also suggested that thymic-derived GCs play a role in the development of thymocytes. However, until now it has not yet been established whether thymic-derived GCs play a role in thymopoiesis in vivo. To investigate this, we conditionally overexpressed the GC receptor (GR) in thymocytes using transgenic mice with a tetracycline-inducible expression system. The influence of systemic GCs was excluded by adrenalectomizing the transgenic mice before the GR induction. Conditional expression of transgenic GR in the thymocytes of adrenalectomized transgenic mice led to a decrease in the thymocyte number. This was associated with increased thymocyte apoptosis. The effect of thymic-derived GCs on the thymocytes was confirmed after transgenic GR induction in a thymic organ culture system. Finally, the GR antagonist RU486 increased thymocyte number in adrenalectomized mice in vivo and prevented a reduction in thymocyte number in thymic organ culture after transgenic GR induction. These observations further confirmed a role for the thymic-derived GCs in regulating thymocyte homeostasis in vivo.

Identification of target genes involved in the antiproliferative effect of glucocorticoids reveals a role for nuclear factor-(kappa)B repression.

Glucocorticoid hormones (GCs) exert an antiproliferative effect on most cells. However, the molecular mechanism is still largely unclear. We investigated the antiproliferative mechanism by GCs in human embryonic kidney 293 cells with stably introduced glucocorticoid receptor (GR) mutants that discriminate between cross-talk with nuclear factor-(kappa)B (NF-(kappa)B) and activator protein-1 signaling, transactivation and transrepression, and antiproliferative vs. non-antiproliferative responses. Using the GR mutants, we here demonstrate a correlation between repression of NF-(kappa)B signaling and antiproliferative response. Gene expression profiling of endogenous genes in cells containing mutant GRs identified a limited number of genes that correlated with the antiproliferative response. This included a GC-mediated up-regulation of the NF-(kappa)B-inhibitory protein I(kappa)B(alpha), in line with repression of NF-(kappa)B signaling being important in the GC-mediated antiproliferative response. Interestingly, the GC-stimulated expression of I(kappa)B(alpha) was a direct effect despite the inability of the GR mutant to transactivate through a GC-responsive element. Selective expression of I(kappa)B(alpha) in human embryonic kidney 293 cells resulted in a decreased percentage of cells in the S/G2/M phase and impaired cell proliferation. These results demonstrate that GC-mediated inhibition of NF-(kappa)B is an important mechanism in the antiproliferative response to GCs.

Different roles for glucocorticoids in thymocyte homeostasis?

Glucocorticoids (GCs) have important immunoregulatory effects on thymocytes and T cells. Ectopic production of GCs has been demonstrated in thymic epithelial cells (TECs) but the role of GCs in thymocyte homeostasis is controversial. Studies in several different mouse models, genetically modified for the GC receptor (GR) expression or function, have demonstrated conflicting results in terms of the effect of the hormone on thymocytes. Here, we summarize these data and suggest that GCs can mediate both positive and negative effects in the organ depending on the local hormonal concentration. Basal GC levels might promote growth of early thymocytes in young mice, and increased levels, generated through a stress reaction, apoptosis in these cells. A gradual loss of GC synthesis in TECs during aging might contribute to thymic involution, a process so far unexplained.

Glucocorticoids delay age-associated thymic involution through directly affecting the thymocytes.

After puberty, the thymus undergoes a dramatic loss in size, resulting in a reduction in the number of thymocytes, a phenomenon termed age-associated thymic involution. The factors regulating this process are poorly understood. We investigated the role of endogenous glucocorticoids (GCs) in this process by studying transgenic mice with increased GC sensitivity restricted to the T-cell lineage due to overexpression of a GC-receptor transgene under the control of the proximal lck promoter. Surprisingly, in these transgenic mice, the age-associated thymic involution did not start until after 6 months of age, demonstrating that endogenous GCs through directly affecting the thymocytes delay the age-associated thymic involution process. The delayed age-associated thymic involution resulted in a significantly higher number of thymocytes in transgenic mice, compared with wild-type mice at 6 months of age or older. The higher number of thymocytes was associated with increased percentage of cycling double-negative and single-positive thymocytes, whereas thymic apoptosis was unaffected. The above effects of GCs were restricted to the thymocytes and were not reflected on the peripheral T cells, in which GCs suppressed the number of peripheral T cells in aged transgenic mice, demonstrating that thymocytes and T cells are differentially regulated by GCs. Furthermore, CD4(+) T cells were more affected than CD8(+) T cells, resulting in a decrease in the CD4/CD8 T-cell ratio. In summary, our results reveal novel biological effects of endogenous GCs on thymic involution and T-cell homeostasis in aged mice.

A pro-apoptotic effect of the CDK inhibitor p57(Kip2) on staurosporine-induced apoptosis in HeLa cells.

Apoptosis, or programmed cell death, is involved in many biological events, including tumorigenesis. Recently, it has been reported that two members of the Cip/Kip family of CDK inhibitors, p21(Cip1) and p27(Kip1), are involved in the regulation of apoptosis. Here, we report that selective expression of the third member in this family, p57(Kip2), potentiated staurosporine-induced apoptosis in HeLa cells. This pro-apoptotic effect was associated with an increased caspase-3 activity. In contrast, glucocorticoid treatment, despite inducing p57(Kip2) expression in HeLa cells, was found to have an inhibitory effect on staurosporine-induced apoptosis. This anti-apoptotic effect of glucocorticoids could be explained by a concomitant increase in Bcl-x(L) expression. The results presented in this study show that p57(Kip2) has a stimulatory effect on apoptosis induced by staurosporine, suggesting a role for p57(Kip2) in the response of tumor cells to cytotoxic drugs.

Effects of altered glucocorticoid sensitivity in the T cell lineage on thymocyte and T cell homeostasis.

The homeostatic regulation that controls total thymocyte and peripheral T-cell numbers is not clearly understood. We describe here a direct hormonal influence of endogenous levels of glucocorticoids (GCs) on thymocyte and peripheral T-cell homeostasis independent of indirect systemic effects of GCs. The results were obtained by generating transgenic mice with an altered GC sensitivity targeted to thymocytes and peripheral T cells by increasing or decreasing glucocorticoid receptor (GR) expression specifically in thymocytes and peripheral T cells. A twofold increase in GC sensitivity resulted in a major decrease in thymocyte number, affecting all subpopulations, although single-positive CD8+ cells were less influenced. In the thymus, this was due to increased apoptosis in the organ, whereas proliferation of thymocyte populations was unaffected. In the periphery, a pronounced reduction in T-cell number was seen, demonstrating an effect of endogenous GCs also on T-cell homeostasis. The effects were confirmed in transgenic mice with reduced GR expression, which showed increased thymocyte and T-cell numbers. Thus, our data demonstrate that physiological GC levels are directly involved in controlling the size of both thymocyte and T-cell pools.