John J. Krolewski

G-protein-independent Activation of Tyk2 by the Platelet-activating Factor Receptor

Platelet-activating factor (PAF) is a potent pro-inflammatory phospholipid with multiple physiological and pathological effects. PAF exerts its activity through a specific heptohelical G-protein coupled receptor, expressed on a variety of cell types, including leukocytes. In this study, we showed that PAF induced a rapid tyrosine phosphorylation of the Tyk2 kinase in the monocytic cell lines U937 and MonoMac-1. PAF-initiated Tyk2 phosphorylation was also observed in COS-7 cells transiently transfected with the human PAF receptor (PAFR) and Tyk2 cDNAs. In addition, we found that Tyk2 co-immunoprecipitated and co-localized with PAFR, independently of ligand binding. Deletion mutants of Tyk2 indicated that the N terminus of the kinase was important for the binding to PAFR. Activation of Tyk2 was followed by a time-dependent 2–4-fold increase in the level of tyrosine phosphorylation of signal transducers and activators of transcription 1 (STAT1), STAT2, and STAT3 and a sustained 2.5-fold increase in STAT5 tyrosine phosphorylation. In MonoMac-1 cells, STAT1 and STAT3 translocated to the nucleus following PAF stimulation, and their translocation in transiently transfected COS-7 cells was shown to be dependent on the presence of Tyk2. In addition, when COS-7 cells were transfected with PAFR and constructs containing PAFR promoter 1, coupled to the luciferase reporter gene, PAF induced a 3.6-fold increase in promoter activation in the presence of Tyk2. Finally, PAFR mutants that could not couple to G-proteins were found to effectively mediate Tyk2 activation and signaling. Taken together, these findings suggest an important role for the Janus kinase/STAT pathway in PAFR signaling, independent of G-proteins, and in the regulation of PAF receptor expression by its ligand.

Cytokine and growth factor receptors in the nucleus: What's up with that?

Signaling via cell surface receptors that are anchored by a single transmembrane domain is a well‐established paradigm. Ligand binding to the extracellular domain of the receptor facilitates receptor dimerization, which juxtaposes the intracellular domains, typically activating intrinsic or associated kinases. Two large families of tyrosine kinase activating receptors have been particularly well characterized: the receptor‐type protein tyrosine kinases and the receptors for the α‐helical cytokines, which activate non‐covalently bound JAK family tyrosine kinases. Despite the well‐established function of these receptors at the cell surface, both intact and cleaved receptors belonging to these families have been repeatedly detected in the nucleus. Furthermore, there is evidence that some of these receptors or receptor fragments function directly in modulating gene transcription. In this essay, I examine how close we are to demonstrating that direct translocation of receptors, or receptor fragments, from the cell surface to the nucleus is a physiologically relevant means of intracellular signaling that can supplant or complement canonical signaling cascades.

In vivo MRI volumetric measurement of prostate regression and growth in mice

AbstractBackground Mouse models for treatment of late-stage prostate cancer are valuable tools, but assessing the extent of growth of the prostate and particularly its regression due to therapeutic intervention or castration is difficult due to the location, small size and interdigitated anatomy of the prostate gland in situ. Temporal monitoring of mouse prostate regression requires multiple animals and examination of histological sections.MethodsInitially, T2-weighted magnetic resonance imaging (MRI) was performed on normal year-old C57/BL6 mice. Individual mice were repeatedly imaged using inhalation anesthesia to establish the reproducibility of the method and to follow hormone manipulation of the prostate volume. Subsequently, MRI fat signal was suppressed using a chemical shift-selective (CHESS) pulse to avoid signal contamination and enhance discrimination of the prostate.ResultsHigh field (7T) MRI provides high resolution (117 × 117 μm in plane), highly reproducible images of the normal mouse prostate. Despite long imaging times, animals can be imaged repeatedly to establish reliability of volume measurements. Prostate volume declines following castration and subsequently returns to normal with androgen administration in the same animal. CHESS imaging allowed discrimination of both the margins of the prostate and the dorsal-lateral lobes of the prostate (DLP) from the ventral lobes (VP). Castration results in a 40% reduction in the volume of the DLP and a 75% reduction in the volume of the VP.ConclusionMRI assessment of the volume of the mouse prostate is precise and reproducible. MRI improves volumetric determination of the extent of regression and monitoring of the same mouse over time during the course of treatment is possible. Since assessing groups of animals at each time point is avoided, this improves the accuracy of the measurement of any manipulation effect and reduces the number of animals required.

Androgen regulation of FLICE-like inhibitory protein gene expression in the rat prostate.

In hope of eventually identifying defects in human prostatic neoplasias that render them insensitive to anti‐androgen therapy, we have examined the regulation of components of ligand‐induced cell death pathways during castration‐induced regression of the prostate. Rat prostates were obtained after surgical castration with or without subsequent androgen replacement. The mRNA levels of genes encoding components of the apoptotic pathway were measured from individual prostates. Whole prostates 1–10 days after castration did not show a significant change in mRNA levels encoding either Fas or FasL, which some studies suggest are necessary for regression to occur. However, the mRNA encoding a catalytically inactive cysteinyl aspartate‐specific protease (caspase) analog, FLICE‐like inhibitor protein (FLIP), decreases during the first day following castration. In the most apoptotically responsive ventral lobe of the rat prostate, the reduction in FLIP mRNA levels is evident within 12 h of castration. The mRNA levels of the principal target of FLIP inhibition, caspase‐8, do not change during the period preceding the onset of detectable DNA fragmentation. Androgen administration to castrated rats reverses prostate regression, and restores FLIP mRNA to normal levels. By acting as an inhibitor of caspase‐8, FLIP may protect prostatic epithelium from apoptosis. Androgen withdrawal, by reducing FLIP mRNA levels, might leave the cells vulnerable to as yet unidentified cell death signals. J. Cell. Physiol. 196: 386–393, 2003.

TNF Is Necessary for Castration-Induced Prostate Regression, Whereas TRAIL and FasL Are Dispensable

TNF, a proinflammatory and immune-regulatory cytokine, is a potent apoptotic stimulus in vitro. However, there have been few examples of a physiologic role for TNF-induced apoptosis in vivo. Here, we describe a novel role for TNF in prostate epithelial cell apoptosis after androgen withdrawal. Employing high-resolution serial magnetic resonance imaging to measure mouse prostate volume changes over time, we demonstrate that the extent of castration-induced prostate regression is significantly reduced in mice null for either the Tnf or Tnfr1 genes but not mice deficient for TNF-related apoptosis-inducing ligand or Fas signaling. Wild-type mice receiving soluble TNF (sTNF) receptor 2 (to bind TNF and block signaling) before castration exhibit an identical reduction of prostate regression. Together, these data indicate that uniquely among known extrinsic death signals, TNF is required for castration-induced prostate regression. Additionally, membrane-bound TNF protein and stromal cell specific TNF mRNA levels increase in rat prostate after castration. This is consistent with a paracrine role for TNF in prostate regression. When injected into the peritoneum of Tnf(-/-) mice at the time of castration, sTNF restores normal levels of prostate regression. However, wild-type mice receiving sTNF in the absence of castration do not exhibit prostate regression, indicating that TNF alone is not sufficient but acts in the context of additional castration-induced signals. These findings support a physiologic role for TNF in prostate regression after androgen withdrawal. Understanding this role may lead to novel therapies for prostate cancer.

Transforming growth factor ß1 induces apoptosis by suppressing FLICE-like inhibitory protein in DU145 prostate epithelial cells

Transforming growth factor ß (TGFß) is a paracrine mediator of prostate epithelial cell apoptosis. In rodents, castration induces production of TGFβ by stromal cells, which leads to apoptosis of epithelial cells. To identify potential mediators of this cell death pathway, we developed a model using DU145 cells, a tumorigenic human prostate epithelial cell line. We discovered that at low density, in low mitogen media, DU145 cells apoptose when treated with TGFβ1. Prior to the onset of death, TGFβ1 treatment downregulated the expression of the caspase inhibitor FLICE‐like inhibitory protein (FLIP), at both the mRNA and protein level, suggesting a causal role between FLIP downregulation and cell death. To confirm the importance of FLIP in TGFβ1‐induced apoptosis, we employed small interfering RNA (siRNA) to silence FLIP expression. Doing so led to apoptosis, which is consistent with the hypothesis that FLIP prevents death in these cells. Furthermore, inhibition of caspase‐8 by siRNA knockdown partially rescued the apoptotic effects of TGFβ1, suggesting a role for death receptor signaling components in TGFß‐mediated death of prostate epithelial cells.

FLICE-Like Inhibitory Protein Blocks Transforming Growth Factor β1–Induced Caspase Activation and Apoptosis in Prostate Epithelial Cells

Androgen withdrawal induces the regression of human prostate cancers, but such cancers eventually become androgen-independent and metastasize. Thus, deciphering the mechanism of androgen withdrawal–induced apoptosis is critical to designing new therapies for prostate cancer. Previously, we showed that in the rat, castration-induced apoptosis is accompanied by a reduction in the expression of the apical caspase inhibitor FLICE-like inhibitory protein (FLIP). To test the functional role of FLIP in inhibiting prostate epithelial cell apoptosis, we employed the rat prostate epithelial cell line NRP-152, which differentiates to a secretory phenotype in a low-mitogen medium and then undergoes apoptosis following the addition of transforming growth factor β1 (TGFβ1), mimicking androgen withdrawal–induced apoptosis. FLIP levels decline with TGFβ1 treatment, suggesting that apoptosis is mediated by caspase-8 and indeed the caspase inhibitor crmA blocks TGFβ1-induced apoptosis. Small interfering RNA–mediated knockdown of FLIP recapitulates and enhances TGFβ1-induced cell death. NRP-152 cells stably transfected with constitutively expressed FLIP were refractory to TGFβ1-induced apoptosis. TGFβ1-induced caspase-3 activity is proportional to the level of cell death and inversely proportional to the level of FLIP expression in various clones. Moreover, neither caspase-3 nor PARP is cleaved in clones expressing high levels of FLIP. Furthermore, insulin, which inhibits differentiation, increases FLIP and inhibits TGFβ-induced death in a FLIP-dependent manner. Although neither Fas-Fc, sTNFRII-Fc, nor DR5-Fc blocked TGFβ1-induced cell death, there is a significant increase in tumor necrosis factor mRNA following TGFβ stimulation, suggesting both an unexpected role for tumor necrosis factor in this model system and the possibility that FLIP blocks another unknown caspase-dependent mediator of apoptosis. (Mol Cancer Res 2008;6(2):231–12)

Intracellular domain of the IFNaR2 interferon receptor subunit mediates transcription via Stat2

We recently demonstrated that IFNaR2, a subunit of the interferon receptor, can be proteolytically cleaved in response to interferon‐alpha and other activators of protein kinase C. Cleavage occurs at multiple sites, via a mechanism similar to that employed by Notch and the Alzheimers precursor protein, and releases the intracellular domain (ICD). In this study, we demonstrate that the IFNaR2 ICD, when fused to the yeast Gal4 DNA binding domain (Gal4DBD) selectively modulates transcription of four different promoters under the control of Gal4 upstream activating sequences. We previously showed that Stat2 binds constitutively to the ICD of IFNaR2, in a manner that is independent of tyrosine phosphorylation. Here, we show that ICD transcriptional modulation is dependent upon the carboxyl‐terminal transactivation domain of Stat2. Specifically, complementing Stat2 deficient cells with wild‐type Stat2 restored the ICD‐mediated transcriptional effects while complementation with a mutant form of Stat2 lacking the transcriptional activation domain (TAD) did not. In addition, mutation of the Stat2 binding site on the ICD reduced the transcriptional activity of the Gal4DBD–ICD. Finally, we demonstrate that the activity of Jak1, a tyrosine kinase also known to bind to IFNaR2, is required for ICD‐mediated transcriptional effects.

Nuclear transit of the intracellular domain of the interferon receptor subunit IFNaR2 requires Stat2 and Irf9

Regulated intramembrane proteolysis (RIP) is the primary signaling mechanism for some receptors, such as Notch and the amyloid precursor protein. In addition, some receptor type tyrosine kinases, such as HER4, are able to signal via both kinase activation and regulated receptor proteolysis. Previously, we showed that the IFNaR2 subunit of the type I interferon receptor can be cleaved in a two step process that resembles RIP and that the IFNaR2 intracellular domain (IFNaR2-ICD) can mediate gene transcription in a Stat2 dependent manner. Here, we demonstrate that IFNaR2-ICD, Stat2 and Irf9 form a ternary complex. Furthermore, Stat2 and Irf9 are required for the nuclear transit of a GFP-linked IFNaR2-ICD construct (GFP-ICD). Additional experiments monitoring the nuclear localization of GFP-ICD demonstrate that Stat2 serves an adaptor role, mediating the interaction between the IFNaR2-ICD and Irf9, while the bipartite nuclear localization signal within Irf9 is the primary determinant driving nuclear transit of the ICD containing complex. Overall, the data suggest that liberation of the IFNaR2-ICD by regulated proteolysis could trigger a novel mechanism for moving the transcription factor Stat2 to the nucleus.