NF-κB-Inducing Kinase Regulates Cyclooxygenase 2 Gene Expression in Macrophages by Phosphorylation of PU.1

May combine two claims.

IκBα degradation occurs in both wild-type and NIK knockdown RAW cells after endotoxin, but is slightly delayed in the knockdown cells (nadir at 30 min vs 15 min).

“Degradation of IB␣ occurred in both the wild-type (Fig. 5A, middle panel, lanes 1-5) and NIK knockdown RAW cells (Fig. 5A, middle panel, lanes 6 -10); however, there this was slightly delayed with a nadir at 30 min in the NIK knockdown RAW cells (Fig. 5A, middle panel, lane 8) compared with 15 min (Fig. 5A, middle panel, lane 2) in the wild-type RAW cells.”

May combine two claims.

At the distal cEts site, activated PU.1 is recruited only at 60 min in wild-type RAW cells and is absent in NIK knockdown cells at that time.

“For the distal cEts site, activated PU.1 was recruited to the COX-2p only at the 60-min time point (Fig. 5C, lane 3) and this was not detected in the NIK knockdown RAW cells at this time point (Fig. 5C, lane 6).”

May combine two claims.

CKII phosphorylates both wild-type PU.1 and the Ser148 mutant.

“In contrast, CKII can phosphorylate either wild type or the mutant PU.1.”

Probably not a substantial claim: may not pass S3 (load-bearing).

The faster-migrating band pulled down by GST-PU.1 is a truncated Myc-NIK fragment missing its C terminus.

“These data (Fig. 1B) show that the lower band that binds to the GST-PU.1 is a C-terminal truncation degradation product of Myc-NIK.”

May combine two claims.

In a 0-6 h ChIP assay, wild-type cells show very slight PU.1 binding to the COX-2 promoter, while NIK knockdown cells show none.

“In this experiment, we detected a very slight binding of PU.1 to the COX-2p in wild-type cells but none was detected in the siRNA NIK”

May combine two claims.

Wild-type RAW cells recruit abundant PU.1 to the proximal cEts site at 30 and 60 min after endotoxin, while NIK knockdown cells show scant binding at those times.

“For the proximal cEts site (Fig. 5A), in response to treatment with endotoxin, abundant PU.1 was recruited to the COX-2p by 30 and 60 min (Fig. 5A, lanes 2 and 3) in the wild-type RAW cells compared with very scant binding to the proximal cEts site at these time points in the NIK knockdown RAW cells (Fig. 5A, lanes 5 and 6).”

May combine two claims.

Immunoprecipitated NIK was unable to phosphorylate the Ser148 mutant PU.1, whereas CKII phosphorylates both wild-type and mutant PU.1.

“the immunoprecipitated NIK was unable to phosphorylate the Ser 148 mutant PU.1 but CKII phosphorylates both wild-type and the mutant PU.1.”

May combine two claims.

CKII has DRB-sensitive kinase action for PU.1 in vitro, but this does not appear to be activated in vivo by endotoxin because NIK knockdown RAW cells with normal CKII levels failed to activate PU.1 to bind to the COX-2 promoter.

“We were able to confirm that CKII has DRB-sensitive kinase action for PU.1 in vitro but this does not seem to be activated in vivo by treatment with endotoxin because the NIK knockdown RAW cells, with normal CKII levels, failed to activate PU.1 to bind to the COX-2p.”

May combine two claims.

Purified myc-NIK had strong in vitro kinase activity for purified GST-PU.1, and this activity and COX-2 protein production are blocked by the nonspecific kinase inhibitor DRB.

“Purified myc-NIK had a strong in vitro kinase activity for purified GST-PU.1, and this activity and production of COX-2 protein is blocked by treatment with a nonspecific kinase inhibitor, 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole.”

May combine two claims.

A stable NIK knockdown macrophage cell line was developed using siRNA, which had an approximately 50% decrease in COX-2 protein production and decreased PGD2 generation, correlated with decreased binding of activated PU.1 to the COX-2 promoter after endotoxin.

“We used short interfering RNA to develop a stable NIK knockdown macrophage cell line that had an ϳ50% decrease in COX-2 protein production and decreased generation of PGD 2 , and this was correlated with decreased binding of activated PU.1 to the COX-2 promoter in response to treatment with endotoxin.”

May combine two claims.

In wild-type RAW cells, PU.1 binding to the COX-2 promoter decreased after 1 h and was undetectable at 6 h.

“After 1 h, PU.1 binding to COX-2p was reduced and was not detected at the 6-h time point in the wild-type cells.”

May combine two claims.

In wild-type RAW cells, PU.1 binding to the COX-2 promoter was very slight at baseline, and no binding was detected in NIK knockdown cells.

“In this experiment, we detected a very slight binding of PU.1 to the COX-2p in wild-type cells but none was detected in the siRNA NIK knockdown cells.”

May combine two claims.

The p38 MAPK pathway and IκBα degradation were not affected by loss of NIK in siRNA-treated RAW 264.7 cells.

“Endotoxin treatment and immunoblot showing that the p38 MAPK pathway and IB␣ degradation were not affected by loss of NIK in siRNA-treated RAW 264.7 cells.”

May combine two claims. The quote only partly states this claim.

Endotoxin treatment did not change immunoreactive NIK levels in either cell type, but NIK levels were greatly reduced in NIK knockdown cells compared with wild-type cells.

“Treatment with endotoxin had no effect on the amount of immunoreactive NIK in the two cell types but, of course, this was greatly reduced in the NIK knockdown RAW cells compared with the wild-type RAW cells.”

May combine two claims.

The NIK-deficient cell line produced less COX-2 in response to endotoxin, and this correlated with decreased PGD2 production.

“The NIK-deficient cell line produced less COX-2 in response to treatment with endotoxin and this was correlated with decreased production of PGD 2 .”

Probably not a substantial claim: may not pass S3 (load-bearing).

CKII can phosphorylate PU.1 in vitro.

“It is already known that RAW cells contain abundant CKII in the cytoplasmic compartment and we confirmed the findings of others that CKII can phosphorylate PU.1 (data not shown).”

Probably not a substantial claim: may not pass S3 (load-bearing).

PU.1 has a DNA-binding domain that recognizes the core sequence 5'-GGAA/T-3'.

“PU.1 is characterized by the presence of a DNA-binding domain that recognizes a core DNA element 5Ј-GGAA/T-3Ј (1-4)”

• cites [1] 1990Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [2] 1993Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [3] 1993Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [4] 1995Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Classified as probably not a substantial claim.

The COX-2 promoter contains binding motifs for PU.1, CREB, NF-κB, and C/EBP-β that contribute to cell-selective COX-2 expression.

“Promoter analysis of the COX-2p shows the presence of several transcription factor-binding motifs, like PU.1, CREB, NF-B, and C/EBP-␤, that result in cell-selective COX-2 gene expression (7).”

• cites [7] 2004Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

NIK mediates lymphotoxin β-induced phosphorylation of RelA at serine 536 and phosphorylates/regulates RelB and RelC.

“NIK has been shown to mediate lymphotoxin ␤-induced phosphorylation of RelA at serine 536 and to phosphorylate and regulate RelB and RelC (21,22).”

• cites [21] 2003Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [22] 2006Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Classified as probably not a substantial claim. May combine two claims.

PU.1 can be phosphorylated at five serine residues (Ser41, Ser45, Ser132, Ser133, Ser148) and serves as an in vitro substrate for CKII.

“PU.1 is potentially phosphorylated on five separate serine residues (Ser 41 , Ser 45 , Ser 132 , Ser 133 , and Ser 148 ) and is an in vitro substrate for casein kinase II (CKII) 3 (5).”

• cites [5] 1997Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

May combine two claims.

NIK is mainly cytoplasmic in unstimulated macrophages and rapidly moves to the nucleus after endotoxin treatment.

“We have reported that NIK predominantly resides in the cytoplasm of unstimulated macrophages and rapidly translocates to the nuclear compartment in response to endotoxin treatment.”

• cites [25] 2006Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

May combine two claims.

Removing NIK leaves endotoxin-induced p38 MAPK and NF-κB signaling unchanged.

“In our experiments, activation of the NF-B and p38 pathways in NIK knockdown macrophages was not affected.”

• Also stated in: § GST-PU.1 is phosphorylated in vitro by Myc-NIK-enriched lysate · hedged

  “These data suggest that loss of NIK does not affect either of these signaling pathways in response to treatment with endotoxin.”

May combine two claims.

NIK is highly selective for PU.1 Ser148, a residue needed for transcriptional activity.

“Thus, NIK is remarkable specific for the Ser 148 residue of PU.1 that is necessary for transcriptional activity.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

NIK is a serine/threonine protein kinase that interacts with TNFR-associated factor 2, IKKα, and IKKβ.

“NF-B-inducing kinase (NIK) is a serine/threonine protein kinase and has been shown to interact with TNFR-associated factor 2, IKK␣, and IKK␤ (19,20)”

• cites [19] 1997Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [20] 2004Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

May combine two claims.

NIK participates directly or indirectly in PU.1 phosphorylation, and its kinase activity does not act on PU.1 mutated at Ser148.

“These experiments indicate that NIK is directly or indirectly involved in phosphorylation of PU.1 and that the kinase activity is not effective in phosphorylating PU.1 that is mutated at the Ser 148 residue.”

Probably not a substantial claim: may not pass S3 (load-bearing).

PU.1 is phosphorylated at Ser41 and Ser142 by Ras-PI3K-dependent ERK, Akt, and p38 MAPK pathways.

“PU.1 is also phosphorylated at Ser 41 and Ser 142 by Ras-PI3K-dependent externally regulated kinase, Akt, and p38 MAPK-mediated pathways (6).”

• cites [6] 2003Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

May combine two claims.

PU.1 binds the COX-2 promoter after endotoxin treatment, leading to COX-2 gene expression.

“We have recently reported (7) that PU.1 binds to the cyclooxygenase 2 promoter (COX-2p) in vitro in response to treatment with endotoxin and this leads to COX-2 gene expression.”

• cites [7] 2004Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Probably not a substantial claim: may not pass S3 (load-bearing).

The NF-κB/Rel family has a central role in inducible expression of many genes involved in inflammation, host defense, cell survival, and proliferation.

“The NF-B/Rel family has a central role in the inducible expression of a large number of genes involved in inflammation, host defense, cell survival, and proliferation (12,13).”

• cites [12] 1999Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [13] 2002Not checked yet

  Not checked in the free run.

Classified as probably not a substantial claim. May combine two claims.

NIK is the specific kinase that phosphorylates PU.1 upon endotoxin stimulation and regulates COX-2 promoter activity for COX-2 expression and PGD2 synthesis.

“Our data are summarized as a schematic depicting our hypothesis (Fig. 7) that NIK is the specific kinase that phosphorylates PU.1 upon endotoxin stimulation and regulates COX-2p activity for COX-2 expression and PGD 2 synthesis.”

• Also stated in: § Discussion · hedged

  “A schematic depicting our proposed hypothesis that NIK is the specific kinase that phosphorylates PU.1 upon endotoxin stimulation.Phosphorylated PU.1 in turn regulates COX-2p activity for COX-2 expression and PGD 2 synthesis.”

• AssumptionRests on an assumption: CKII is a cytosolic kinase.
• AssumptionRests on an assumption: Activation of PU.1 by phosphorylation is confined to the nuclear compartment.

May combine two claims.

Involvement of CKII in PU.1 phosphorylation was ruled out, although immunoreactive CKII was not detected in anti-Myc immunoprecipitates for the Myc-NIK fusion molecule.

“We have ruled out involvement by CKII, although we were unable to detect immunoreactive CKII in the anti-Myc immunoprecipitates for the Myc-NIK fusion molecule.”

May combine two claims.

NIK knockdown RAW cells showed reduced COX-2 protein production and enzymatic activity compared with wild-type cells, and this reduction correlated with decreased binding of activated PU.1 to the COX-2 promoter.

“Thus, there was reduced production of COX-2 protein production and enzymatic activity in NIK knockdown RAW cells compared with wild-type RAW cells and this correlated with decreased binding of activated PU.1 to the COX-2p.”

Classified as probably not a substantial claim. May combine two claims.

In many cases, phosphorylation of PU.1 is required for regulation of gene transcription, such as COX-2 and TLR4, and it modulates interactions with cofactors, other transcription factors, trans-activating activity, or DNA-binding activity.

“In many cases, phosphorylation of PU.1 is necessary for regulation of gene transcription (6,7,33,34), like COX-2 and TLR4 (7,11), which in turn modulates interaction with cofactors, other transcription factors, trans-activating activity, or DNA-binding activity.”

• cites [6] 2003Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [7] 2004Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [33] 1998Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [34] 2001Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [11] 2005Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Probably not a substantial claim: may not pass S3 (load-bearing).

PU.1 interacts with multiple transcription factors, including IFN regulatory factor 4, c-JUN, and GATA-1, leading to combinatorial transcriptional regulation.

“PU.1 interacts with many transcription factors, including IFN regulatory factor 4 (NF-EM5/PIP, c-JUN, and GATA-1 (9,32), to result in combinatorial transcriptional regulation of gene expression.”

• cites [9] 1993Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [32] 1999Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Probably not a substantial claim: may not pass S3 (load-bearing).

Blocking COX-2 specifically in macrophages could disturb eicosanoid balance when several cell sources are stimulated together.

“Specific interdiction in macrophage COX-2 gene expression could disrupt the complicated balance of eicosanoids when multiple cellular sources are concomitantly stimulated.”

Classified as probably not a substantial claim. May combine two claims.

Little is known about the specific kinase that phosphorylates PU.1, although several reports indicate CKII has in vitro kinase activity for PU.1.

“Little is known about the specific kinase that phosphorylates PU.1, although there are several reports that CKII has in vitro kinase activity for PU.1 (5,9).”

• cites [5] 1997Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• cites [9] 1993Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

• AssumptionRests on an assumption: There are no in vivo experimental data clearly showing that CKII phosphorylates PU.1 in response to endotoxin.

Probably not a substantial claim: may not pass S3 (load-bearing).

The study cannot totally exclude the possibility that immunoprecipitated NIK acts with an unknown kinase or cofactor present in the immunoprecipitated complex.

“In these studies, we are not able to totally exclude the possibility that immunoprecipitated NIK acts in conjunction with an unknown kinase or cofactor that might be present in the immunoprecipitated complex.”

May combine two claims.

Since nonmyeloid cells do not express PU.1, NIK-dependent PU.1 phosphorylation might permit selective COX-2 inhibition in macrophages while sparing structural and mesenchymal cells.

“Because PU.1 is not expressed by nonmyeloid cell types, NIK-mediated phosphorylation of PU.1 could allow a highly selective inhibition of COX-2 in macrophages that could potentially spare COX-2 gene expression in structural and mesenchymal cell types.”

• AssumptionRests on an assumption: Nonmyeloid cells do not produce PU.1.

May combine two claims.

NIK immunoprecipitates were tested for CKII contamination, and CKII antibodies failed to detect CKII in the immunoprecipitates.

“We tested the NIK immunoprecipitates for CKII contamination, but CKII Abs failed to detect any CKII presence in our immunoprecipitates.”

May combine two claims.

NIK affects COX-2 gene expression through two independent mechanisms: phosphorylation of PU.1, which has a combinatorial role in regulating COX-2 gene transcription, and phosphorylation of IKKα, which has a role in histone H3 phosphorylation and regulation of chromosomal structure and COX-2 promoter availability for transcription factor binding.

“Thus, NIK affects COX-2 gene expression through two independent mechanisms, phosphorylation of PU.1, which has a combinatorial role in regulating COX-2 gene transcription, and phosphorylation of IKK␣, which has a role in phosphorylation of histone H3 and regulation of chromosomal structure and the availability of the COX-2p for binding of transcription factors.”

May combine two claims.

NIK translocates to the nucleus in response to endotoxin treatment of RAW cells and plays an important role in regulating PU.1 phosphorylation and its binding to the COX-2 gene promoter.

“In summary, our study suggests that NIK translocates to the nucleus in response to endotoxin treatment of RAW cells and plays an important role in regulating PU.1 phosphorylation and its binding to the COX-2 gene promoter.”

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

The final COX-2 product depends on cell type: macrophages and mast cells make proinflammatory PGD2, epithelial cells make counterinflammatory PGE2, platelets make vasoconstrictor thromboxane, and endothelial cells make vasodilator prostacyclin.

“The exact end product of COX-2 is dependent on the cellular source.Macrophages and mast cells produce PGD 2 which is proinflammatory, epithelial cells produced PGE 2 which is counterinflammatory, platelets produce thromboxane, which is a vasoconstrictor, and endothelial cells produce prostacyclin, which is a vasodilator.”

• cites [30] 2006Not matched

  Couldn’t be matched to a paper in Crossref, so it can’t be checked.

Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

Growth factor stimulation increases CKII activity, but studies have not demonstrated CKII translocation from cytoplasm to nucleus in response to endotoxin, and no studies have demonstrated a protein-protein interaction between CKII and PU.1 or NIK.

“Cells that are stimulated with growth factors have been shown to have increased CKII activity, but these studies failed to demonstrate translocation of CKII from the cytoplasm to the nuclear compartment in response to treatment with endotoxin, and no studies have demonstrated a protein-protein interaction between CKII and PU.1 or NIK.”