Pno1 Expression Pattern in WT Mice According to in situ Hybridization
Based on the exposure time (optimal time: 4 days) necessary to produce X-Ray film autoradiogram, Pno1 mRNA belonged to a class of low abundant mouse mRNAs.
At mid-gestation embryo on day 9 (e9), Pno1 signals in most tissues, including the rudimental brain (MB: midbrain; FB: forebrain; NT: neural tube) and cardiovascular system (H: heart; AA: aortic arch), were relatively high, as illustrated in . After this stage and at subsequent gestation ages, density and spatial hybridization patterns underwent substantial change. The large cerebral Pno1-positive area was reduced to the periventricular band region on e11 and e13 (). Pno1 mRNA level in the liver (Li) was moderate to high on e13 (), but dropped significantly on e16 (). In contrast, Pno1 mRNA signals became perceptible in the kidneys (K) and Th, starting on e16 (–IVD). Peak of Pno1 expression was observed in developing striated muscles (M) and intermingled adipose tissue on e16 (). Heart ventricles on e19 transiently displayed Pno1 mRNA levels () higher than on e9 (panel H in ). Around parturition (e19 and post-natal day 1 [p1]), Pno1 mRNA was clearly present in the immune system such as the thymus (Th), the digestive system such as the submaxillary gland (SMax), stomach (St), intestine (Int) and colon (descending) (CD)), peripheral nervous system (dorsal root ganglia (DRG), sympathetic ganglia (SG)), and skin (Sk) (). However, Pno1 mRNA signals were only slightly above background in the remaining tissues.
ISH analysis of Pno1 expression during ontogeny.
The Pno1 expression pattern on p10 remained similar to that on p1 (). Several particular brain regions, such as the olfactory lobe (OL), olfactory neuroepithelium (ONE) and hippocampus (Hip), manifested high expression levels; signals in the molars (Mol), trigeminal ganglion (TriG), Int and bone marrow (BM) were also elevated; the spleen displayed moderate expression ().
In adulthood, Pno1 expression was noted in teeth, such as the incisors (Inc) (), vertebrate bone marrow (Vb) () and testes (Ts) (). Pno1 expression in adult Sk seemed to be less abundant than in p10 Sk (comparative data not included). In lymphoid organs, the thymus continued to present high expression levels (), while expression in the spleen remained moderate or low (). Expression in the thymus occurred mainly in the cortex (). Adult mouse BM cells displayed Pno1 signals both in x-ray film autoradiography () and emulsion autoradiography ( and III). BM in the Vb cavity showed labelled islands containing cells with small diameters (likely hematopoietic cells) in the midst of a meshwork of unlabelled connective tissue cells ( and III). The adrenal glands that are composed of amine- and peptide-producing medulla (Me), and steroid hormone-producing cortex (Cx) displayed Pno1 mRNA labelling in both regions, but predominantly in Cx (, II and III).
Pno1 expression in adult tissues according to ISH.
Generation of Pno1 Gene KO Mice
To understand Pno1 function, we generated Pno1 gene KO mice. The targeting strategy is depicted in . With the 3′ end probe, the WT allele after BamHI digestion gave an 18.6-kb band on Southern blotting, and the KO allele, a 7.7-kb band (). Germline transmission was confirmed by Southern blotting of tail DNA, and WT and HET mice were thus identified (). PCR was undertaken for routine genotyping of ear DNA. WT samples presented a 548-bp band, and HET samples, a 224-bp band ().
To ascertain if Pno1 gene deletion affected its expression, we measured Pno1 mRNA levels in different tissues by RT-qPCR. As shown in , HET samples from the Li, Th and Spl presented lower Pno1 mRNA levels compared to WT samples. Pno1 was previously selected for study because it was inducible after T cell activation (data not reported). We activated T cells with solid phase anti-CD3 and anti-CD28 monoclonal antibodies (mAbs), and quantified Pno1 mRNA levels at different time points (1, 2, 6, 12 and 24 h after the initiation of culture). As expected, WT T cells presented augmented Pno1 mRNA levels between 6 and 24 h after activation. On the other hand, HET T cells only up-regulated Pno1 mRNA to about half of that in WT T cells (). The data in confirmed the gene deletion of Pno1 in HET mice. They also indicated that Pno1 expression was gene copy number-dependent, as HET cells only expressed half of Pno1 at the mRNA level compared to WT cells.
Pno1 KO is Lethal in Embryos
We failed to generate any live Pno1 KO mice. Systemic tracking of fetus genotype in different gestation stages revealed that KO fetuses could only be found at e3.5 but not e6.5 (). Therefore, the embryos must have died between e3.5 and e6.5. We then cultured e1.5 embryos from HET male and HET female mating to observe their development in vitro. As depicted in , up to e2.5, WT, HET and KO development seemed to be comparable. However, after e3.5, KO embryos stopped developing, while WT and HET embryos proceeded normally at this stage and beyond, indicating that Pno1 is vital in embryonic development.
Genotypic analysis of embryos from PNO1+/− × PNO1+/− mating.
In vitro development of WT, HET and KO embryos.
No Detectable Anomalies in the Immune System of Pno1 HET Mice
As Pno1 was prominently expressed in the thymus and was up-regulated in T cells upon their activation, we set out to investigate whether a lack of Pno1 causes immune system abnormalities. HET mice, which expressed Pno1 at about 50% the normal Pno1 level, were used for this purpose because no KO mice could be produced. There were no apparent lymphoid organ anomalies in HET mice in terms of size, weight and colour (data not reported). T, B, CD4 and CD8 sub-populations in the spleen and lymph nodes (LN) of HET mice were comparable to those in WT mice, as were thymocyte sub-populations, such as CD4CD8 double-negative, CD4CD8 double-positive, CD4 single-positive and CD8 single-positive cells ().
Sub-populations of lymphocytes in lymphoid organs and T cell function of Pno1 HET mice.
Although HET T cells could only express Pno1 at about 50% the normal Pno1 level upon activation by anti-TCR mAb on solid phase, they proliferated as well as WT T cells (). Activation markers, such as CD25 and CD69, were up-regulated in HET CD4 and CD8 T cells comparably to their WT counterparts (). Therefore, it seems that 50% of normal Pno1 expression is sufficient to maintain T cell development and function.
We also assessed B cell subpopulations in the spleen and peritoneal cavity of WT and HET mice. In the spleen of WT and HET mice, there was no consistent difference in terms of the percentage of B220+CD21hiCD23lo/− mantel zone B cells (), or the percentage of B220+CD21int/hiCD23hi follicular B cells (). For B cells of the WT and HET mouse peritoneal exudates, there was no consistent difference in terms of the percentage of B220+CD23−CD5+IgMhi B1a cells (), B220+CD23−CD5−IgMhi B1b () and B220+CD23+IgMint/hi B2 cells (). Spleen B cells from WT and HET mice proliferated comparably upon anti-IgM plus IL-4, LPS plus IL-4, anti-CD40 plus IL-4, or anti-CD40 plus LPS stimulation (). The upregulation of B cell activation markers CD86 and CD80 in WT and HET spleen B cells 24 h after the above-described stimulation showed no apparent difference neither (). We next examined the percentage of IgD−IgM−CD138+B220lo/− isotype-switched plasmablast/plasma cells in the draining lymph nodes of WT and HET mice 21 days after immununization with chick type II collagen, but no significant difference was found (). The serum collagen-specific Ab levels in these WT and HET mice 21 days after the chick type II collagen immunization showed no significant difference (). Therefore, it seems that a 50% reduction of Pno1 level does not affect the B cells functions in vitro and in vivo.
B cell development and function in HET mice.
Normal Proteasome Activity in HET Tissues
Because Pno1 is involved in proteasome maturation, according to studies in yeasts, we questioned whether reduced Pno1 expression in HET mice leads to decreased proteasome activity in cells. We tested 3 major proteasome protease activities (i.e., chymotrypsin-like, trypsin-like and caspase-like proteases, but the caspase-like proteases activity is not detectable) in splenocytes and thymocytes of HET mice, and discerned that they were comparable to those of WT mice (). We also measured the protein level of one subunit (β5) of the proteasome complex in the lungs, spleens, thymuses, livers and kidneys of HET mice, and found that it was comparable to that of WT tissues (). Taken together, these data suggest that a 50% reduction of Pno1 expression does not elicit abnormal proteasome levels or activities.
Proteasome activities WT and HET tissues.
Generation of Pno1 Tg Mice
Since we could not obtain live Pno1 KO mice, and since HET mice manifested no apparent anomalies in general and in immune system and proteasome activities in particular, we wondered whether Pno1 over-expression in mice would reveal some phenotype. We generated Tg mice with β-actin promoter-driven Pno1 expression. The plasmid construct is shown in . Tail DNA was routinely genotyped by PCR, and Tg mice presented a 552-bp band (). In Tg organs, such as the lung, kidney, thymus and spleen, Pno1 mRNA expression was universally heightened, although to different degrees (). We also tested chymotrypsin-like and trypsin-like protease activities in the Tg thymus and spleen, and found that they were similar to those of their WT counterparts ().
Pno1 Tg Mice Manifest no Apparent Immune System Anomalies
Pno1 Tg mice were fertile and had no gross anomalies. Their immune organs, the thymus, LN and Spleen, were of normal size and cellularity (data not reported). T and B cell populations and CD4 and CD8 T cell populations in the spleen and LN were comparable to those of WT mice (). In the Tg thymus, the different thymic sub-populations (e.g., CD4CD8 double-negative, CD4CD8 double-positive, CD4 single-positive and CD8 single-positive cells) were similar to those in the WT thmus (). Pno1 over-expression did not compromise T cell proliferation caused by solid phase anti-CD3 mAb stimulation (). Tg CD4 and CD8 T cells up-regulated their activation markers CD25 and CD69 in comparison to their WT counterparts upon activation (). Therefore, excessive Pno1 expression does not seem to affect proteasome activity.
Sub-populations of lymphocytes in lymphoid organs and T cell function of Pno1 Tg mice.
We also assessed B cell subpopulations in the spleen and peritoneal cavity of WT and Tg mice. In the spleen of WT and Tg mice, there was no consistent difference in terms of the percentage of B220+CD21hiCD23lo/− mantel zone B cells (), or the percentage of B220+CD21int/hiCD23hi follicular B cells (). For B cells of the WT and Tg mouse peritoneal exudates, there was no consistent difference in terms of the percentage of B220+CD23-CD5+IgMhi B1a cells (), B220+CD23-CD5-IgMhi B1b () and B220+CD23+IgMint/hi B2 cells (). Spleen B cells from WT and Tg mice proliferated comparably upon anti-IgM plus IL-4, LPS plus IL-4, anti-CD40 plus IL-4, or anti-CD40 plus LPS stimulation (). The upregulation of B cell activation markers CD86 and CD80 in WT and Tg spleen B cells 24 h after the above-described stimulation showed no apparent difference neither (). We next examined the percentage of IgD-IgM-CD138+B220lo/− isotype-switched plasmablast/plasma cells in the draining lymph nodes of WT and Tg mice 21 days after immununization with chick type II collagen, but not significant difference was found (). The serum collagen-specific Ab levels in these WT and Tg mice 21 days after the chick type II collagen immunization showed no significant difference ().
B cell development and function in HET mice.
The results from this section suggest that a normal endogenous Pno1 expression level is sufficient for the tested T cell functions. Pno1 over-expression does not confer additional advantages.
Pno1 Occurs in Complexes Larger than 26S Proteasomes
We wondered whether Pno1 is present in proteasome complexes in mammalian cells. For this purpose, we stably transfected L cells with a Pno1-expressing construct, pCEP-Pno1, in which Pno1 was fused with 3 copies of HA tags at the N-terminus. The transfected cells expressed greatly-enhanced Pno1 mRNA levels (), but their proteasome activity remained similar to that of empty vector-transfected cells (). L cell lysates were fractionated with glycerol density gradients, and the chymotrypsin-like proteasome activity of each fraction was quantified. As seen in , there were 2 peaks of enzymatic activity at fractions 13 and 17, corresponding to 20S and 26S proteasomes, respectively. Western blotting showed that the proteasome β5 subunit was present from fractions 13 to 21, with trace amounts found up to fraction 23 (), confirming that proteasome complexes (20S and 26S) were in these fractions, which contained 2 peaks of proteasome activities (). Interestingly, Pno1, as detected by anti-HA Ab, made a major appearance, starting from fraction 13 and ending at fraction 25, with trace amounts occurring until fraction 27 (). Its location did not correspond exactly to that of 20S or 26S proteasomes based on both proteasome activity and β5 location, but obviously resided in fractions larger than 26S.
Pno1 is associated with macromolecules with a sedimentation rate greater than 26S.
Is Pno1 then a part of ribosomes? We assessed small and large ribosome subunits in glycerol gradient fractions by anti-S6 and anti-L7 mAb Western blotting (, respectively). S6 signals appeared from fraction 17 and ended at fraction 27, consistent with the size of the small 40S ribosome subunit. L7 signals presented from fraction 25 until fraction 27, consistent with the size of large 60S ribosome subunits. Fractions containing the small subunit overlapped with but were not identical to those of Pno1.
The results of this sedimentation study indicate that Pno1 does not co-localize exactly with proteasomes, small or large ribosome subunits, but seems to exist in complexes with sedimentation rates higher than 26S, probably between 30S to 40S, employing a low ribosome sedimentation rate as marker.