Deficiency of myotubularin-related protein 14 influences body weight,metabolism, and inflammation in an age-dependent manner

Background

Myotubularin-related protein 14 (MTMR14) is a novel phosphoinositide phosphatase with roles in the maintenance of normal muscle performance, autophagy, and aging in mice. Our initial pilot study demonstrated that MTMR14 knock out (KO) mice gain weight earlier than their wild-type (WT) littermates, which suggests that this gene may also be involved in metabolism regulation.

Results

The present study evaluated the role of MTMR14 in the development of aging-associated obesity. We found that aged MTMR14 KO mice fed a normal chow diet exhibited increased serum triglyceride, total cholesterol, and glucose levels compared to age-matched WT controls. Lipid accumulation was also increased in aged KO mice. Several inflammatory cytokines and adipokines were dramatically dysregulated in the metabolic tissues of aged MTMR14 KO mice compared to control mice. Circulating inflammatory cytokines were significantly elevated and plasma adipokine levels were abnormally regulated in aged MTMR14 KO mice. These data suggest that MTMR14 deficiency caused a late-onset inflammation and metabolic dysfunction. Further study demonstrated that this exacerbated metabolic dysfunction and inflammation may be regulated by the phosphoinositide 3 kinase/protein kinase B and extracellular signal-regulated protein kinase signaling pathways.

Conclusions

Our current research suggests that MTMR14 deletion induces overweight and adult obesity accompanied by chronic inflammation in an age-dependent manner.

     

Mice lacking myotubularin-related protein 14 show accelerated high-fat diet-induced lipid accumulation and inflammation

The phosphoinositide phosphatase, myotubularin-related protein 14 (MTMR14), has been reported to play an important role in the regulation of muscle performance, autophagy, and aging in mice. We previously showed that MTMR14-knockout (KO) mice gain weight earlier than their wild-type (WT) littermates even on a normal chow diet (NCD), suggesting that this gene might also be involved in regulating metabolism. In the present study, we evaluated the effect of MTMR14 deficiency on high-fat diet (HFD)-induced obesity, lipid accumulation, metabolic disorders, and inflammation in WT and MTMR14-KO mice fed with NCD or HFD. To this end, MTMR14-KO mice fed with HFD showed significantly increased body weight, blood glucose levels, serum triglyceride (TG) levels, and total cholesterol (TC) levels as compared to their age-matched WT control. Additionally, lipid accumulation also increased in the KO mice. Simultaneously, the expression of metabolism-associated genes (Glut4, adiponectin, and leptin) was different in the liver, muscle, and fatty tissue of MTMR14-KO mice fed with HFD. More importantly, the expression of several inflammation-associated genes (TNF-α, IL-6, IL-1β, and MCP-1) dramatically increased in the liver, muscle, and fatty tissue of MTMR14-KO mice relative to control. Taken together, these results suggest that MTMR14 deficiency accelerates HFD-induced metabolic dysfunction and inflammation. Furthermore, the results showed that exacerbated metabolic dysfunction and inflammation may be regulated via the PI3K/Akt and ERK signaling pathways.     

RNase L Induces Autophagy via c-Jun N-terminal Kinase and Double-stranded RNA-dependent Protein Kinase Signaling Pathways

Autophagy is a tightly regulated mechanism that mediates sequestration, degradation, and recycling of cellular proteins, organelles, and pathogens. Several proteins associated with autophagy regulate host responses to viral infections. Ribonuclease L (RNase L) is activated during viral infections and cleaves cellular and viral single-stranded RNAs, including rRNAs in ribosomes. Here we demonstrate that direct activation of RNase L coordinates the activation of c-Jun N-terminal kinase (JNK) and double-stranded RNA-dependent protein kinase (PKR) to induce autophagy with hallmarks as accumulation of autophagic vacuoles, p62(SQSTM1) degradation and conversion of Microtubule-associated Protein Light Chain 3-I (LC3-I) to LC3-II. Accordingly, treatment of cells with pharmacological inhibitors of JNK or PKR and mouse embryonic fibroblasts (MEFs) lacking JNK1/2 or PKR showed reduced autophagy levels. Furthermore, RNase L-induced JNK activity promoted Bcl-2 phosphorylation, disrupted the Beclin1-Bcl-2 complex and stimulated autophagy. Viral infection with Encephalomyocarditis virus (EMCV) or Sendai virus led to higher levels of autophagy in wild-type (WT) MEFs compared with RNase L knock out (KO) MEFs. Inhibition of RNase L-induced autophagy using Bafilomycin A1 or 3-methyladenine suppressed viral growth in initial stages; in later stages autophagy promoted viral replication dampening the antiviral effect. Induction of autophagy by activated RNase L is independent of the paracrine effects of interferon (IFN). Our findings suggest a novel role of RNase L in inducing autophagy affecting the outcomes of viral pathogenesis.     

Knockout of ATG5 leads to malignant cell transformation and resistance to Src family kinase inhibitor PP2

Autophagy can either promote or inhibit cell death in different cellular contexts. In this study, we investigated the role of autophagy in ATG5 knockout (KO) cell line established using CRISPR/Cas9 system. In ATG5 KO cells, RT‐PCR and immunoblot of LC3 confirmed the functional gene knockout. We found that knockout of ATG5 significantly increased proliferation of NIH 3T3 cells. In particular, autophagy deficiency enhanced susceptibility to cellular transformation as determined by an in vitro clonogenic survival assay and a soft agar colony formation assay. We also found that ATG5 KO cells had a greater migration ability as compared to wild‐type (WT) cells. Moreover, ATG5 KO cells were more resistant to treatment with a Src family tyrosine kinase inhibitor (PP2) than WT cells were. Cyto‐ID Green autophagy assay revealed that PP2 failed to induce autophagy in ATG5 KO cells. PP2 treatment decreased the percentage of cells in the S and G₂/M phases among WT cells but had no effect on cell cycle distribution of ATG5 KO cells, which showed a high percentage of cells in the S and G₂/M phases. Additionally, the proportion of apoptotic cells significantly decreased after treatment of ATG5 KO cells with PP2 in comparison with WT cells. We found that expression levels of p53 were much higher in ATG5 KO cells. The ATG5 KO seems to lead to compensatory upregulation of the p53 protein because of a decreased apoptosis rate. Taken together, our results suggest that autophagy deficiency can lead to malignant cell transformation and resistance to PP2.     

GADD34 suppresses wound healing by upregulating expression of myosin IIA

Wound healing consists of sequential steps of tissue repair, and cell migration is particularly important. In order to analyze the potential function of growth arrest and DNA damage inducible protein 34 (GADD34) in tissue repair, we performed in vitro and in vivo wound healing experiments. In an in vitro scratch assay, GADD34 knockout (KO) mouse embryonic fibroblasts (MEFs) had higher migration rates than did wild type (WT) MEFs. Furthermore, the rate of wound closure was faster in GADD34 KO MEFs than in WT MEFs. Using in vivo punch biopsy assays, GADD34 KO mice had accelerated wound healing compared to WT mice. WT mice expressed higher amounts of myosin IIA in migrating macrophages and myofibroblasts than did GADD34 KO mice. These results indicate that GADD34 negatively regulates cell migration in wound healing via expression of myosin IIA.     

Suppression of autophagy by FIP200 deletion impairs DNA damage repair and increases cell death upon treatments with anticancer agents

Autophagy is a lysosomal bulk degradation process for intracellular protein and organelles. FIP200 (200 kDa FAK-family interacting protein) is an essential component of mammalian autophagy that is implicated in breast cancer in recent studies. Here we show that inactivation of FIP200 resulted in deficient repair of DNA damage induced by ionizing radiation and anticancer agents in mouse embryonic fibroblasts (MEF). The persistent DNA damage correlated to increased apoptosis and reduced survival of FIP200 knockout (KO) MEFs after treatments with camptothecin (CPT), a topoisomerase I inhibitor and chemotherapeutic agent. Reexpression of FIP200 in FIP200 KO MEFs restored both efficient DNA damage repair and cell survival. Furthermore, knockdown of the increased p62 expression in FIP200 KO MEFs rescued the impaired DNA damage repair and CPT-induced cell death. In contrast, treatment of cells with N-acetyl cysteine did not affect these defects in FIP200 KO MEFs. Finally, FIP200 KO MEFs also showed deficient DNA damage repair and increased cell death compared with control MEFs, when treated with etoposide, a topoisomerase II inhibitor and another anticancer agent. Together, these results identify a new function for FIP200 in the regulation of DNA damage response and cell survival through its activity in autophagy and suggest the possibility of FIP200 or other autophagy proteins as a potential target for treatment to enhance the efficiency of cancer therapy using DNA damage-inducing agents.     

Dynamics of the degradation of ubiquitinated proteins by proteasomes and autophagy: association with sequestosome 1/p62

Proteotoxicity resulting from accumulation of damaged/unwanted proteins contributes prominently to cellular aging and neurodegeneration. Proteasomal removal of these proteins upon covalent polyubiquitination is highly regulated. Recent reports proposed a role for autophagy in clearance of diffuse ubiquitinated proteins delivered by p62/SQSTM1. Here, we compared the turnover dynamics of endogenous ubiquitinated proteins by proteasomes and autophagy by assessing the effect of their inhibitors. Autophagy inhibitors bafilomycin A1, ammonium chloride, and 3-methyladenine failed to increase ubiquitinated protein levels. The proteasome inhibitor epoxomicin raised ubiquitinated protein levels at least 3-fold higher than the lysosomotropic agent chloroquine. These trends were observed in SK-N-SH cells under serum or serum-free conditions and in WT or Atg5(-/-) mouse embryonic fibroblasts (MEFs). Notably, chloroquine considerably inhibited proteasomes in SK-N-SH cells and MEFs. In these cells, elevation of p62/SQSTM1 was greater upon proteasome inhibition than with all autophagy inhibitors tested and was reduced in Atg5(-/-) MEFs. With epoxomicin, soluble p62/SQSTM1 associated with proteasomes and p62/SQSTM1 aggregates contained inactive proteasomes, ubiquitinated proteins, and autophagosomes. Prolonged autophagy inhibition (96 h) failed to elevate ubiquitinated proteins in rat cortical neurons, although epoxomicin did. Moreover, prolonged autophagy inhibition in cortical neurons markedly increased p62/SQSTM1, supporting its degradation mainly by autophagy and not by proteasomes. In conclusion, we clearly demonstrate that pharmacologic or genetic inhibition of autophagy fails to elevate ubiquitinated proteins unless the proteasome is affected. We also provide strong evidence that p62/SQSTM1 associates with proteasomes and that autophagy degrades p62/SQSTM1. Overall, the function of p62/SQSTM1 in the proteasomal pathway and autophagy requires further elucidation.     

INK4a deletion results in improved kidney regeneration and decreased capillary rarefaction after ischemia-reperfusion injury

The molecular mechanisms that lead to tubular atrophy, capillary loss, and fibrosis following acute kidney injury are not very clear but may involve cell cycle inhibition by increased expression of cyclin kinase inhibitors. The INK4a/ARF locus encodes overlapping genes for two proteins, a cyclin kinase inhibitor, p16(INK4a), and a p53 stabilizer, p19(ARF), from independent promoters. To determine if decreased INK4a gene expression results in improved kidney regeneration, INK4a knockout (KO) and wild-type (WT) mice were subjected to ischemia-reperfusion injury (IRI). p16(INK4a) and p19(ARF) levels were increased markedly in WT mice at 1-28 days after injury. Kidneys were examined to determine the localization and levels of p16(INK4a), apoptosis, cell proliferation, and capillary rarefaction. KO mice displayed decreased tubular cell apoptosis, increased cell proliferation, and lower creatinine levels after injury. KO mice had significantly higher capillary density compared with WT mice at 14-42 days after IRI. Plasma granulocyte colony-stimulating factor (G-CSF) increased after ischemia in both WT and KO mice and was elevated markedly in KO compared with WT mice. KO kidney digests contained higher counts of Gr-1(+)/Cd11b(+) myeloid cells by flow cytometry. KO mice treated with a Gr-1-depleting antibody displayed reduced vascular endothelial growth factor mRNA, plasma G-CSF, and capillary density, and an increase in serum creatinine and medullary myofibroblasts, compared with untreated KO mice 14 days after ischemia. The anti-angiogenic effect of Gr-1 depletion in KO mice was confirmed by Matrigel angiogenesis assays. These results suggest that the absence of p16(INK4a) and p19(ARF) following IRI has a protective effect on the kidney through improved epithelial and microvascular repair, in part by enhancing the mobilization of myeloid cells into the kidney.     

Caveolin-1 is required for fatty acid translocase (FAT/CD36) localization and function at the plasma membrane of mouse embryonic fibroblasts

Several lines of evidence suggest that lipid rafts are involved in cellular fatty acid uptake and influence fatty acid translocase (FAT/CD36) function. However, it remains unknown whether caveolae, a specialized raft type, are required for this mechanism. Here, we show that wild-type (WT) mouse embryonic fibroblasts (MEFs) and caveolin-1 knockout (KO) MEFs, which are devoid of caveolae, have comparable overall expression of FAT/CD36 protein but altered subcellular FAT/CD36 localization and function. In WT MEFs, FAT/CD36 was isolated with both lipid raft enriched detergent-resistant membranes (DRMs) and detergent-soluble membranes (DSMs), whereas in cav-1 KO cells it was exclusively associated with DSMs. Subcellular fractionation demonstrated that FAT/CD36 in WT MEFs was localized intracellularly and at the plasma membrane level while in cav-1 KO MEFs it was absent from the plasma membrane. This mistargeting of FAT/CD36 in cav-1 KO cells resulted in reduced fatty acid uptake compared to WT controls. Adenoviral expression of caveolin-1 in KO MEFs induced caveolae formation, redirection of FAT/CD36 to the plasma membrane and rescue of fatty acid uptake. In conclusion, our data provide evidence that caveolin-1 is necessary to target FAT/CD36 to the plasma membrane. Caveolin-1 may influence fatty acid uptake by regulating surface availability of FAT/CD36.     

MAPK14/p38α confers irinotecan resistance to TP53-defective cells by inducing survival autophagy

Recently we have shown that the mitogen-activated protein kinase (MAPK) MAPK14/p38α is involved in resistance of colon cancer cells to camptothecin-related drugs. Here we further investigated the cellular mechanisms involved in such drug resistance and showed that, in HCT116 human colorectal adenocarcinoma cells in which TP53 was genetically ablated (HCT116-TP53KO), overexpression of constitutively active MAPK14/p38α decreases cell sensitivity to SN-38 (the active metabolite of irinotecan), inhibits cell proliferation and induces survival-autophagy. Since autophagy is known to facilitate cancer cell resistance to chemotherapy and radiation treatment, we then investigated the relationship between MAPK14/p38α, autophagy and resistance to irinotecan. We demonstrated that induction of autophagy by SN38 is dependent on MAPK14/p38α activation. Finally, we showed that inhibition of MAPK14/p38α or autophagy both sensitizes HCT116-TP53KO cells to drug therapy. Our data proved that the two effects are interrelated, since the role of autophagy in drug resistance required the MAPK14/p38α. Our results highlight the existence of a new mechanism of resistance to camptothecin-related drugs: upon SN38 induction, MAPK14/p38α is activated and triggers survival-promoting autophagy to protect tumor cells against the cytotoxic effects of the drug. Colon cancer cells could thus be sensitized to drug therapy by inhibiting either MAPK14/p38 or autophagy.     

Follistatin promotes adipocyte differentiation,browning, and energy metabolism

Follistatin (Fst) functions to bind and neutralize the activity of members of the transforming growth factor-β superfamily. Fst has a well-established role in skeletal muscle, but we detected significant Fst expression levels in interscapular brown and subcutaneous white adipose tissue, and further investigated its role in adipocyte biology. Fst expression was induced during adipogenic differentiation of mouse brown preadipocytes and mouse embryonic fibroblasts (MEFs) as well as in cold-induced brown adipose tissue from mice. In differentiated MEFs from Fst KO mice, the induction of brown adipocyte proteins including uncoupling protein 1, PR domain containing 16, and PPAR gamma coactivator-1α was attenuated, but could be rescued by treatment with recombinant FST. Furthermore, Fst enhanced thermogenic gene expression in differentiated mouse brown adipocytes and MEF cultures from both WT and Fst KO groups, suggesting that Fst produced by adipocytes may act in a paracrine manner. Our microarray gene expression profiling of WT and Fst KO MEFs during adipogenic differentiation identified several genes implicated in lipid and energy metabolism that were significantly downregulated in Fst KO MEFs. Furthermore, Fst treatment significantly increases cellular respiration in Fst-deficient cells. Our results implicate a novel role of Fst in the induction of brown adipocyte character and regulation of energy metabolism.     

Parkin deficiency increases the resistance of midbrain neurons and glia to mild proteasome inhibition: the role of autophagy and glutathione homeostasis

Parkin mutations in humans produce parkinsonism whose pathogenesis is related to impaired protein degradation, increased free radicals and abnormal neurotransmitter release. In this study, we have investigated whether partial proteasomal inhibition by epoxomicin, an ubiquitin proteasomal system (UPS) irreversible inhibitor, further aggravates the cellular effects of parkin suppression in midbrain neurons and glia. We observed that parkin null (PK‐KO) midbrain neuronal cultures are resistant to epoxomicin‐induced cell death. This resistance is due to increased GSH and DJ‐1 protein levels in PK‐KO mice. The treatment with epoxomicin increases, in wild type (WT) cultures, the pro‐apoptotic Bax/Bcl‐2 ratio, the phosphorylation of tau, and the levels of chaperones heat‐shock protein 70 and C‐terminal Hsc‐interacting protein, but none of these effects took place in epoxomicin‐treated PK‐KO cultures. Poly‐ubiquitinated proteins increased more in WT than in PK‐KO‐treated neuronal cultures. Parkin accumulated in WT neuronal cultures treated with epoxomicin. Markers of autophagy, such as LC3II/I, were increased in naïve PK‐KO cultures, and further increased after treatment with epoxomicin, implying that the blockade of the proteasome in PK‐KO neurons triggers the enhancement of autophagy. The treatment with l ‐buthionine‐S,R‐sulfoximine and the inhibition of autophagy, however, reverted the increase resistance to epoxomicin of the PK‐KO cultures. We also found that PK‐KO glial cells, stressed by growth in defined medium and depleted of GSH, were more susceptible to epoxomicin induced cell death than WT glia treated similarly. This susceptibility was linked to reduced GSH levels and less heat‐shock protein 70 response, and to activation of p‐serine/threonine kinase protein signaling pathway as well as to increased poly‐ubiquitinated proteins. These data suggest that mild UPS inhibition is compensated by other mechanisms in PK‐KO midbrain neurons. However the depletion of GSH, as happens in stressed glia, suppresses the protection against UPS inhibition‐induced cell death. Furthermore, GSH inhibition regulated differentially UPS activity and in old PK‐KO mice, which have depletion of GSH, UPS activity is decreased in comparison with that of old‐WT.     

Rapamycin inhibits the secretory phenotype of senescent cells by a Nrf2‐independent mechanism

Senescent cells contribute to age‐related pathology and loss of function, and their selective removal improves physiological function and extends longevity. Rapamycin, an inhibitor of mTOR, inhibits cell senescence in vitro and increases longevity in several species. Nrf2 levels have been shown to decrease with aging and silencing Nrf2 gene induces premature senescence. Therefore, we explored whether Nrf2 is involved in the mechanism by which rapamycin delays cell senescence. In wild‐type (WT) mouse fibroblasts, rapamycin increased the levels of Nrf2, and this correlates with the activation of autophagy and a reduction in the induction of cell senescence, as measured by SA‐β‐galactosidase (β‐gal) staining, senescence‐associated secretory phenotype (SASP), and p16 and p21 molecular markers. In Nrf2KO fibroblasts, however, rapamycin still decreased β‐gal staining and the SASP, but rapamycin did not activate the autophagy pathway or decrease p16 and p21 levels. These observations were further confirmed in vivo using Nrf2KO mice, where rapamycin treatment led to a decrease in β‐gal staining and pro‐inflammatory cytokines in serum and fat tissue; however, p16 levels were not significantly decreased in fat tissue. Consistent with literature demonstrating that the Stat3 pathway is linked to the production of SASP, we found that rapamycin decreased activation of the Stat3 pathway in cells or tissue samples from both WT and Nrf2KO mice. Our data thus suggest that cell senescence is a complex process that involves at least two arms, and rapamycin uses Nrf2 to regulate cell cycle arrest, but not the production of SASP.     

MAPK14/p38α confers irinotecan resistance to TP53-defective cells by inducing survival autophagy

Recently we have shown that the mitogen-activated protein kinase (MAPK) MAPK14/p38α is involved in resistance of colon cancer cells to camptothecin-related drugs. Here we further investigated the cellular mechanisms involved in such drug resistance and showed that, in HCT116 human colorectal adenocarcinoma cells in which TP53 was genetically ablated (HCT116-TP53KO), overexpression of constitutively active MAPK14/p38α decreases cell sensitivity to SN-38 (the active metabolite of irinotecan), inhibits cell proliferation and induces survival-autophagy. Since autophagy is known to facilitate cancer cell resistance to chemotherapy and radiation treatment, we then investigated the relationship between MAPK14/p38α, autophagy and resistance to irinotecan. We demonstrated that induction of autophagy by SN38 is dependent on MAPK14/p38α activation. Finally, we showed that inhibition of MAPK14/p38α or autophagy both sensitizes HCT116-TP53KO cells to drug therapy. Our data proved that the two effects are interrelated, since the role of autophagy in drug resistance required the MAPK14/p38α. Our results highlight the existence of a new mechanism of resistance to camptothecin-related drugs: upon SN38 induction, MAPK14/p38α is activated and triggers survival-promoting autophagy to protect tumor cells against the cytotoxic effects of the drug. Colon cancer cells could thus be sensitized to drug therapy by inhibiting either MAPK14/p38 or autophagy.     

The reductase NCB5OR is responsive to the redox status in beta-cells and is not involved in the ER stress response

The novel reductase NCB5OR (NADPH cytochrome b5 oxidoreductase) resides in the ER (endoplasmic reticulum) and may protect cells against ER stress. Levels of BiP (immunoglobulin heavy-chain-binding protein), CHOP (CCAAT/enhancer-binding protein homologous protein) and XBP-1 (X-box-binding protein-1) did not differ in WT (wild-type) and KO (Ncb5or-null) tissues or MEFs (mouse embryonic fibroblasts), and XBP-1 remained unspliced. MEFs treated with inducers of ER stress demonstrated no change in Ncb5or expression and expression of ER-stress-induced genes was not enhanced. Induction of ER stress in beta-cell lines did not change Ncb5or expression or promoter activity. Transfection with Ncb5or-specific siRNA (small interfering RNA) yielded similar results. Microarray analysis of mRNA from islets and liver of WT and KO animals revealed no significant changes in ER-stress-response genes. Induction of oxidative stress in betaTC3 cells did not alter Ncb5or mRNA levels or promoter activity. However, KO islets were more sensitive to streptozotocin when compared with WT islets. MEFs incubated with nitric oxide donors showed no difference in cell viability or levels of nitrite produced. No significant differences in mRNA expression of antioxidant enzymes were observed when comparing WT and KO tissues; however, microarray analysis of islets indicated slightly enhanced expression of some antioxidant enzymes in the KO islets. Short-term tBHQ (t-butylhydroquinone) treatment increased Ncb5or promoter activity, although longer incubation times yielded a dose-dependent decrease in activity. This response appears to be due to a consensus ARE (antioxidant-response element) present in the Ncb5or promoter. In summary, NCB5OR does not appear to be involved in ER stress, although it may be involved in maintaining or regulating the redox status in beta-cells.     

Cell-cycle inhibitors p27Kip1 and p21Cip1 regulate murine Sertoli cell proliferation

Thyroid hormone inhibits neonatal Sertoli cell proliferation and recent results have shown that thyroid hormone upregulates cyclin-dependent kinase inhibitors (CDKIs) p27Kip1 and p21Cip1 (also known as CDKN1B and CDKN1A, respectively) in neonatal Sertoli cells. This suggests that these CDKIs, which negatively regulate the cell cycle, could be critical in Sertoli cell proliferation. Consistent with this hypothesis, mice lacking p27Kip1 develop testicular organomegaly, but Sertoli cell numbers have not been determined. Likewise, effects of loss of p21Cip1 or both p27 and p21 on Sertoli cell number and testicular development were unknown. To determine if p27 and/or p21 regulate Sertoli cell proliferation, we measured Sertoli cell proliferation at Postnatal Day 16 and testis weight, Sertoli cell number, and daily sperm production (DSP) in 4-mo-old wild-type (WT), p21 knockout (p21KO), p27 knockout (p27KO), and p27/p21 double-knockout (DBKO) mice. Testis weights were increased 27%, 42%, and 86% in adult p21KO, p27KO, and DBKO mice, respectively, compared with WT. Sertoli cell number also was increased 48%, 126%, and 126% in p21KO, p27KO, and DBKO mice, respectively, versus WT. DSP in p21KO, p27KO, and DBKO testes also showed significant increases compared with WT mice. Although DSP was increased, there were increased spermatogenic defects observed in both p27KO and DBKO mice compared with WT. These data indicate that both p27 and p21 play an inhibitory role in regulating adult Sertoli cell number such that loss of either CDKI produces primary increases in Sertoli cell number and secondary increases in DSP and testis weight. Furthermore, loss of both CDKIs causes additive effects on DSP and testis weight, suggesting a central role for these CDKIs in testis development.