Podocytes are lost from glomeruli before completing apoptosis.

Although apoptosis of podocytes has been widely reported in in vitro studies, it has been less frequently and less definitively documented in in vivo situations. To investigate this discrepancy, we analyzed the dying process of podocytes in vitro and in vivo using LMB2, a human (h)CD25-directed immunotoxin. LMB2 induced cell death within 2 days in 56.8 ± 13.6% of cultured podocytes expressing hCD25 in a caspase-3, Bak1, and Bax-dependent manner. LMB2 induced typical apoptotic features, including TUNEL staining and fragmented nuclei without lactate dehydrogenase leakage. In vivo, LMB2 effectively eliminated hCD25-expressing podocytes in NEP25 mice. Podocytes injured by LMB2 were occasionally stained for cleaved caspase-3 and cleaved lamin A but never for TUNEL. Urinary sediment contained TUNEL-positive podocytes. To examine the effect of glomerular filtration, we performed unilateral ureteral obstruction in NEP25 mice treated with LMB2 1 day before euthanasia. In the obstructed kidney, glomeruli contained significantly more cleaved lamin A-positive podocytes than those in the contralateral kidney (50.1 ± 5.4% vs. 29.3 ± 4.1%, P < 0.001). To further examine the dying process without glomerular filtration, we treated kidney organoids generated from nephron progenitor cells of NEP25 mice with LMB2. Podocytes showed TUNEL staining and nuclear fragmentation. These results indicate that on activation of apoptotic caspases, podocytes are detached and lost in the urine before nuclear fragmentation and that the physical force of glomerular filtration facilitates detachment. This phenomenon may be the reason why definitive apoptosis is not observed in podocytes in vivo.NEW & NOTEWORTHY This report clarifies why morphologically definitive apoptosis is not observed in podocytes in vivo. When caspase-3 is activated in podocytes, these cells are immediately detached from the glomerulus and lost in the urine before DNA fragmentation occurs. Detachment is facilitated by glomerular filtration. This phenomenon explains why podocytes in vivo rarely show TUNEL staining and never apoptotic bodies.

Sperm associated antigen 4 promotes SREBP1-mediated de novo lipogenesis via interaction with lamin A/C and contributes to tumor progression in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a highly malignant tumor and its progression is associated with altered lipid metabolism in precancerous lesions, such as non-alcoholic fatty liver disease. Here, we identified sperm associated antigen 4 (SPAG4), and explored its oncogenic role in HCC progression. Database analysis and immunohistochemistry indicated increased level of SPAG4 in HCC tissues which was of prognostic value. Gain/loss-of-function experiments showed that SPAG4 exerted oncogenic roles in HCC growth both in vitro and in vivo. RNA sequencing revealed activation of a lipogenic state and SREBP1-mediated pathway following SPAG4 overexpression. Mechanistically, the N-terminal region of SPAG4 bound to lamin A/C, which increased SREBP1 expression, nuclear translocation, and transcriptional activity. Treatment with orlistat, a lipid synthesis inhibitor, reversed SPAG4-mediated oncogenic effects, and its efficacy varied with SPAG4 level. The effect of orlistat was further amplified when combined with sorafenib in tumor xenograft mouse models. Our study provides evidence that SPAG4 mediates HCC progression by affecting lipid metabolism. Administration of orlistat combined with sorafenib reverses SPAG4-mediated oncogenesis in HCC cells and ectopic xenograft tumors in mice, suggesting that this pathway represents a potential target for HCC treatment.

Progerin-Induced Replication Stress Facilitates Premature Senescence in Hutchinson-Gilford Progeria Syndrome.

Hutchinson-Gilford progeria syndrome (HGPS) is caused by a mutation in LMNA that produces an aberrant lamin A protein, progerin. The accumulation of progerin in HGPS cells leads to an aberrant nuclear morphology, genetic instability, and p53-dependent premature senescence. How p53 is activated in response to progerin production is unknown. Here we show that young cycling HGPS fibroblasts exhibit chronic DNA damage, primarily in S phase, as well as delayed replication fork progression. We demonstrate that progerin binds to PCNA, altering its distribution away from replicating DNA in HGPS cells, leading to γH2AX formation, ATR activation, and RPA Ser33 phosphorylation. Unlike normal human cells that can be immortalized by enforced expression of telomerase alone, immortalization of HGPS cells requires telomerase expression and p53 repression. In addition, we show that the DNA damage response in HGPS cells does not originate from eroded telomeres. Together, these results establish that progerin interferes with the coordination of essential DNA replication factors, causing replication stress, and is the primary signal for p53 activation leading to premature senescence in HGPS. Furthermore, this damage response is shown to be independent of progerin farnesylation, implying that unprocessed lamin A alone causes replication stress.

Remodelling of the nuclear lamina and nucleoskeleton is required for skeletal muscle differentiation in vitro.

Changes in the expression and distribution of nuclear lamins were investigated during C2C12 myoblast differentiation. The expression of most lamins was unchanged during myogenesis. By contrast, lamin-B2 expression increased and LAP2alpha expression decreased twofold. These changes were correlated with reduced solubility and redistribution of A-type lamins. When C2C12 myoblasts were transfected with a lamin-A mutant that causes autosomal dominant Emery-Dreifuss muscular dystrophy (AD-EDMD), the mutant protein accumulated in the nucleoplasm and exerted dominant influences over endogenous lamins. Myoblasts transfected with wild-type lamins differentiated, albeit more slowly, whereas myoblasts transfected with mutant lamins failed to differentiate. Myoblast differentiation requires dephosphorylation of the retinoblastoma protein Rb. During myogenesis, Rb was rapidly and progressively dephosphorylated. Underphosphorylated Rb formed complexes with LAP2alpha in proliferating myoblasts and postmitotic myoblasts. In myoblasts transfected with the mutant lamins, this complex was disrupted. These data suggest that remodelling of the nucleoskeleton is necessary for skeletal-muscle differentiation and for correct regulation of Rb pathways.

Interleukin-1beta and TNF-alpha act in synergy to inhibit longitudinal growth in fetal rat metatarsal bones.

UNLABELLED: We hypothesized that pro-inflammatory cytokines can act locally in the growth plate to impair longitudinal growth. In a model of cultured fetal rat metatarsal bones, we found that IL-1beta and TNF-alpha act in synergy to inhibit longitudinal growth, an effect linked to decreased proliferation and increased apoptosis of growth plate chondrocytes. IGF-I could partially reverse all these effects. INTRODUCTION: Children with chronic inflammatory conditions, such as Crohn's disease or rheumatoid arthritis, experience impaired longitudinal growth. The inflammatory process itself, which includes upregulation of the pro-inflammatory cytokines interleukin (IL)-1beta, IL-6, and TNF-alpha, is believed to be at least partly responsible for the poor growth in these patients. This study aimed to clarify whether these cytokines can act locally in the growth plate to suppress longitudinal growth and whether any negative effects can be reversed by insulin-like growth factor-I (IGF-I). MATERIALS AND METHODS: The effects of cytokines on longitudinal bone growth were studied in fetal (day E20) rat metatarsal bones kept in culture. After a 7-day culture, the bones were sectioned, and chondrocyte proliferation was assessed by bromodeoxyuridine (BrdU) incorporation and apoptosis by TUNEL. RESULTS: When added separately, IL-1beta and TNF-alpha impaired longitudinal bone growth only at a high concentration (100 ng/ml each; p < 0.05 versus control). In contrast, when added in combination, IL-1beta and TNF-alpha potently inhibited growth at far lower concentrations (from 3 ng/ml each; p < 0.001 versus control) and also decreased chondrocyte proliferation and increased apoptosis. Growth failure induced by the combination of IL-1beta and TNF-alpha (10 ng/ml each) could be counteracted by anti-IL-1beta (100 ng/ml; p < 0.001), anti-TNF-alpha (100 ng/ml; p < 0.001), or IGF-I (100 ng/ml; p < 0.01). IL-6 did not affect longitudinal growth even when added in combination with IL-1beta or TNF-alpha (10 ng/ml each). CONCLUSIONS: We show that IL-1beta and TNF-alpha act in synergy to locally suppress longitudinal growth, an effect that can be partially reversed by IGF-I. Although growth hormone (GH)/IGF-I may improve longitudinal growth in children with chronic inflammatory diseases, our results suggest that the inflammatory process itself must be targeted to achieve normal growth.