Renoprotective effect of berberine via regulating the PGE2‐EP1‐Gαq‐Ca2+ signalling pathway in glomerular mesangial cells of diabetic rats

G‐protein coupled receptor‐mediated pathogenesis is of great importance in the development of diabetic complications, but the detailed mechanisms have not yet been clarified. Therefore, we aimed to explore the roles of the prostaglandin E2 receptor 1 (EP1)‐mediated signalling pathway and develop a corresponding treatment for diabetic nephropathy (DN). To create the DN model, rats fed a high‐fat and high‐glucose diet were injected with a single dose of streptozotocin (35 mg/kg, i.p.). Then, rats were either treated or not with berberine (100 mg/kg per day, i.g., 8 weeks). Cells were isolated from the renal cortex and cultured in high‐sugar medium with 20% foetal bovine serum. Prostaglandin E₂ (PGE₂) levels were determined by ELISA, and cells were identified by fluorescence immunoassay. We measured the biochemical characteristics and observed morphological changes by periodic‐acid‐Schiff staining. The expression of the EP1 receptor and the roles of GRK2 and β‐arrestin2 were identified using western blotting and flow cytometry. Downstream proteins were detected by western blot, while molecular changes were assessed by ELISA and laser confocal scanning microscopy. Berberine not only improved the majority of biochemical and renal functional parameters but also improved the histopathological alterations. A significant increase in PGE₂ level, EP1 membrane expression and Gαq expression, and concentration of Ca²⁺ were observed, accompanied by increased GRK2 and β‐arrestin2 levels soon afterwards. Berberine decreased the abnormal concentration of Ca²⁺, the increased levels of PGE₂, the high expression of EP1 and Gαq and suppressed the proliferation of mesangial cells. The EP1 receptor, a critical therapeutic target of the signalling pathway, contributed to mesangial cell abnormalities, which are linked to renal injury in DN. The observed renoprotective effects of berberine via regulating the PGE₂‐EP1‐Gαq‐Ca²⁺ signalling pathway indicating that berberine could be a promising anti‐DN medicine in the future.     

Research progress of endothelial‐mesenchymal transition in diabetic kidney disease

Renal fibrosis is an important pathological feature of diabetic kidney disease (DKD), manifested as tubular interstitial fibrosis, tubular atrophy, glomerulosclerosis and damage to the normal structure of the kidney. Renal fibrosis can eventually develop into renal failure. A better understanding of renal fibrosis in DKD is needed due to clinical limitations of current anti‐fibrotic drugs in terms of effectiveness, cost‐effectiveness and side effects. Fibrosis is characterized by local excessive deposition of extracellular matrix, which is derived from activated myofibroblasts to increase its production or specific tissue inhibitors of metalloproteinases to reduce its degradation. In recent years, endothelial‐mesenchymal transition (EndMT) has gradually integrated into the pathogenesis of fibrosis. In animal models of diabetic kidney disease, it has been found that EndMT is involved in the formation of renal fibrosis and multiple signalling pathways such as TGF‐β signalling pathway, Wnt signalling pathway and non‐coding RNA network participate in the regulation of EndMT during fibrosis. Here, we mainly review EndMT regulation and targeted therapy of renal fibrosis in DKD.     

Berberine regulates mesangial cell proliferation and cell cycle to attenuate diabetic nephropathy through the PI3K/Akt/AS160/GLUT1 signalling pathway

High glucose (HG) is one of the basic factors of diabetic nephropathy (DN), which leads to high morbidity and disability. During DN, the expression of glomerular glucose transporter 1 (GLUT1) increases, but the relationship between HG and GLUT1 is unclear. Glomerular mesangial cells (GMCs) have multiple roles in HG‐induced DN. Here, we report prominent glomerular dysfunction, especially GMC abnormalities, in DN mice, which is closely related to GLUT1 alteration. In vivo studies have shown that BBR can alleviate pathological changes and abnormal renal function indicators of DN mice. In vitro, BBR (30, 60 and 90 μmol/L) not only increased the proportion of G1 phase cells but also reduced the proportion of S phase cells under HG conditions at different times. BBR (60 μmol/L) significantly reduced the expression of PI3K‐p85, p‐Akt, p‐AS160, membrane‐bound GLUT1 and cyclin D1, but had almost no effect on total protein. Furthermore, BBR significantly declined the glucose uptake and retarded cyclin D1‐mediated GMC cell cycle arrest in the G1 phase. This study demonstrated that BBR can inhibit the development of DN, which may be due to BBR inhibiting the PI3K/Akt/AS160/GLUT1 signalling pathway to regulate HG‐induced abnormal GMC proliferation and the cell cycle, supporting BBR as a potential therapeutic drug for DN.     

MDM2 is implicated in high‐glucose‐induced podocyte mitotic catastrophe via Notch1 signalling

Podocyte injury and depletion are essential events involved in the pathogenesis of diabetic nephropathy (DN). As a terminally differentiated cell, podocyte is restricted in ‘post‐mitosis’ state and unable to regenerate. Re‐entering mitotic phase will cause podocyte disastrous death which is defined as mitotic catastrophe (MC). Murine double minute 2 (MDM2), a cell cycle regulator, is widely expressed in renal resident cells including podocytes. Here, we explore whether MDM2 is involved in podocyte MC during hyperglycaemia. We found aberrant mitotic podocytes with multi‐nucleation in DN patients. In vitro, cultured podocytes treated by high glucose (HG) also showed an up‐regulation of mitotic markers and abnormal mitotic status, accompanied by elevated expression of MDM2. HG exposure forced podocytes to enter into S phase and bypass G2/M checkpoint with enhanced expression of Ki67, cyclin B1, Aurora B and p‐H3. Genetic deletion of MDM2 partly reversed HG‐induced mitotic phase re‐entering of podocytes. Moreover, HG‐induced podocyte injury was alleviated by MDM2 knocking down but not by nutlin‐3a, an inhibitor of MDM2‐p53 interaction. Interestingly, knocking down MDM2 or MDM2 overexpression showed inhibition or activation of Notch1 signalling, respectively. In addition, genetic silencing of Notch1 prevented HG‐mediated podocyte MC. In conclusion, high glucose up‐regulates MDM2 expression and leads to podocyte MC. Notch1 signalling is an essential downstream pathway of MDM2 in mediating HG‐induced MC in podocytes.     

Down-regulation of SLC35C1 induces colon cancer through over-activating Wnt pathway

The canonical Wnt signalling pathway is a critical pathway involved in the proliferation of cells. It has been well-established that it plays the central role during colorectal carcinogenesis and development. Yet the exact molecular mechanism of how the canonical Wnt pathway is fine-tuned remains elusive. We found that SLC35C1, a GDP-fucose transporter, negatively regulates the Wnt signalling pathway. We show here that SLC35C1 is reduced in all colon cancer by both immunohistochemistry images and TCGA data, whereas β-catenin is increased. Down-regulation of SLC35C1 is also detected by real-time PCR in stage 3 and stage 4 colorectal cancer tissues. Moreover, analysing the TCGA database with cBioPortal reveals the negative correlation of SLC35C1 mRNA level to the expression of β-catenin. Reduced SLC35C1 significantly promotes cell proliferation and colony formation of HEK293 cells. Meanwhile, in HEK293 cells silencing SLC35C1 activates canonical Wnt pathway, whereas overexpressing SLC35C1 inhibits it. Consistently, the reduction of SLC35C1 in HEK293 cells also elevated the mRNA level of Wnt target genes C-myc, Axin2 and Cyclin D1, as well as the secretion of Wnt3a. In conclusion, we identified SLC35C1 as a negative regulator of the Wnt signalling pathway in colon cancer. Decreased SLC35C1 may cause over-activation of Wnt signalling in colorectal cancer.     

Wnt5a promotes renal tubular inflammation in diabetic nephropathy by binding to CD146 through noncanonical Wnt signaling

Immune and inflammatory factors have emerged as key pathophysiological mechanisms in the progression of diabetic renal injury. Noncanonical Wnt5a signaling plays an essential role in obesity- or diabetes-induced metabolic dysfunction and inflammation, but its explicit molecular mechanisms and biological function in diabetic nephropathy (DN) remain unknown. In this study, we found that the expression of Wnt5a and CD146 in the kidney and the level of soluble form of CD146 (sCD146) in serum and urine samples were upregulated in DN patients compared to controls, and this alteration was correlated with the inflammatory process and progression of renal impairment. Blocking the activation of Wnt5a signaling with the Wnt5a antagonist Box5 prevented JNK phosphorylation and high glucose-induced inflammatory responses in db/db mice and high glucose-treated HK-2 cells. Similar effects were observed by silencing Wnt5a with small-interfering RNA (siRNA) in cultured HK-2 cells. Knockdown of CD146 blocked Wnt5a-induced expression of proinflammatory cytokines and activation of JNK, which suggests that CD146 is essential for the activation of the Wnt5a pathway. Finally, we confirmed that Wnt5a directly interacted with CD146 to activate noncanonical Wnt signaling in HK-2 cells. Taken together, our findings suggest that by directly binding to CD146, Wnt5a-induced noncanonical signaling is a contributing mechanism for renal tubular inflammation in diabetic nephropathy. The concentration of sCD146 in serum and urine could be a potential biomarker to predict renal outcomes in DN patients.Subject terms: Kidney diseases, Inflammation     

Association analysis of canonical Wnt signalling genes in diabetic nephropathy

Aims/Hypothesis

Several studies have provided compelling evidence implicating the Wnt signalling pathway in the pathogenesis of diabetic nephropathy. Gene expression profiles associated with renal fibrosis have been attenuated through Wnt pathway modulation in model systems implicating Wnt pathway members as potential therapeutic targets for the treatment of diabetic nephropathy. We assessed tag and potentially functional single nucleotide polymorphisms (SNPs; n = 31) in four key Wnt pathway genes (CTNNB1, AXIN2, LRP5 and LRP6) for association with diabetic nephropathy using a case-control design.

Methods

SNPs were genotyped using Sequenom or Taqman technologies in 1351 individuals with type 1 diabetes (651 cases with nephropathy and 700 controls without nephropathy). Cases and controls were white and recruited from the UK and Ireland. Association analyses were performed using PLINK, to compare allele and haplotype frequencies in cases and controls. Adjustment for multiple testing was performed by permutation testing.

Results

Following logistic regression analysis adjusted by collection centre, duration of T1D, and average HbA1c as covariates, a single SNP in LRP6 (rs1337791) was significantly associated with DN (OR = 0.74; CI: 0.57–0.97; P = 0.028), although this was not maintained following correction for multiple testing. Three additional SNPs (rs2075241 in LRP6; rs3736228 and rs491347 both in LRP5) were marginally associated with diabetic nephropathy, but none of the associations were replicated in an independent dataset. Haplotype and subgroup analysis (according to duration of diabetes, and end-stage renal disease) also failed to reveal an association with diabetic nephropathy.

Conclusions/Interpretation

Our results suggest that analysed common variants in CTNNB1, AXIN2, LRP5 and LRP6 are not strongly associated with diabetic nephropathy in type 1 diabetes among white individuals. Our findings, however, cannot entirely exclude these genes or other members of the Wnt pathway, from involvement in the pathogenesis of diabetic nephropathy as our study had limited power to detect variants with small effect size.     

Dapper-1 is essential for Wnt5a induced cardiomyocyte hypertrophy by regulating the Wnt/PCP pathway

The Wnt signaling pathway was identified as crucial mediator of cardiomyocyte hypertrophy. In this study we found that activation of non-canonical Wnt signaling by Wnt5a stimulates protein synthesis and enlargement of cardiomyocyte surface area. These hypertrophic features were inhibited in Dapper-1 (Dpr1) depleted cells. On the molecular level, we observed inhibition of the non-canonical Wnt/planar-cell-polarity (PCP) pathway denoted by reduction of c-jun-n-terminal-kinase (JNK) phosphorylation. Upstream of JNK, increased protein levels of the Wnt/PCP trans-membrane receptor van-Gogh-like-2 (Vangl2) were observed along with an enrichment of Vangl2 in perinuclear located vesicles. The findings suggest that Dpr1 is essential for execution of the Wnt/PCP pathway and regulation of the Vangl2/JNK axis. Depletion of Dpr1 inhibits non-canonical Wnt signaling induced cardiomyocyte hypertrophy by blocking Wnt/PCP signaling.     

Adipose-Derived Mesenchymal Stem Cells Transplantation Alleviates Renal Injury in Streptozotocin-Induced Diabetic Nephropathy

Previous studies have illustrated that bone marrow-derived mesenchymal stem cell (BMMSC) transplantation has therapeutic effects on diabetes and can prevent mice from renal damage and diabetic nephropathy (DN). Moreover, adipose-derived MSCs possess similar characteristics to BMMSCs. We investigated the effect of ADMSC transplantation on streptozotocin (STZ)-induced renal injury. Diabetes was induced in rats by STZ injection. After ADMSC treatment, renal histological changes and cell apoptosis were evaluated as were the expression of apoptosis-related proteins, Wnt/β-catenin pathway members, and klotho levels. We found that ADMSCs improved renal histological changes. Next, NRK-52E cells were exposed to normal glucose (NG; 5.5 mM glucose plus 24.5 mM mannitol)/high glucose (HG) or ADMSCs, and then measured for changes in the aforementioned proteins. Similarly, changes in these proteins were also determined following transient transfection of klotho siRNA. We found that both ADMSC transplantation and co-incubation reduced the rate of cellular apoptosis, decreased Bax and Wnt/β-catenin levels, and elevated Bcl-2 and klotho levels. Interestingly, klotho knockdown reversed the effects of ADMSCs on the expression of apoptosis-related proteins and Wnt/β-catenin pathway members. Taken together, ADMSCs transplantation might attenuate renal injury in DN via activating klotho and inhibiting the Wnt/β-catenin pathway. This study may provide evidence for the treatment of DN using ADMSCs.     

Inhibition of MAPK‐mediated ACE expression by compound C66 prevents STZ‐induced diabetic nephropathy

A range of in vitro, experimental and clinical intervention studies have implicated an important role for hyperglycaemia‐induced activation of the renin‐angiotensin system (RAS) in the development and progression of diabetic nephropathy (DN). Blockade of RAS by angiotensin converting enzyme (ACE) inhibitors is an effective strategy in treating diabetic kidney diseases. However, few studies demonstrate the mechanism by which hyperglycaemia up‐regulates the expression of ACE gene. Our previous studies have identified a novel curcumin analogue, (2E,6E)‐2,6‐bis(2‐(trifluoromethyl)benzylidene)cyclohexanone (C66), which could inhibit the high glucose (HG)‐induced phosphorylation of mitogen‐activated protein kinases in mouse macrophages. In this study, we found that the renal protection of C66 in diabetic mice was associated with mitogen‐activated protein kinase (MAPK) inactivation and ACE/angiotensin II (Ang II) down‐regulation. Generally, MAPKs have been considered as a downstream signalling of Ang II and a mediator for Ang II‐induced pathophysiological actions. However, using C66 and specific inhibitors as small molecule probes, in vitro experiments demonstrate that the MAPK signalling pathway regulates ACE expression under HG stimulation, which contributes to renal Ang II activation and the development of DN. This study indicates that C66 is a potential candidate of DN therapeutic agents, and more importantly, that reduction in ACE expression by MAPKs inhibition seems to be an alternative strategy for the treatment of DN.     

Zebrafish Dapper1 and Dapper2 play distinct roles in Wnt-mediated developmental processes

Wnt signaling pathways in vertebrates use the phosphoprotein Dishevelled (Dvl). The cellular responses to Wnt signaling may in part be modulated by Dvl-associated proteins, including Dapper (Dpr). We have cloned and characterized the zebrafish Dpr paralogs Dpr1 and Dpr2. Loss-of-function studies reveal that endogenous Dpr1 but not Dpr2 is required to enhance Wnt/beta-catenin activity in zebrafish embryos that are hypomorphic for Wnt8. Conversely, Dpr2 but not Dpr1 is required for normal convergence extension movements in embryos that are hypomorphic for Stbm or Wnt11, supporting a functional interaction of Dpr2 with Wnt/Ca2+-PCP signaling. In gain-of-function experiments, Dpr1 but not Dpr2 induces Wnt/beta-catenin target genes. Dpr1 synergizes with zebrafish Dvl2, and with the Dvl-interacting kinases CK1epsilon, Par1 and CK2, in activating target genes. We conclude that two Dvl-associated paralogs, Dpr1 and Dpr2, participate in distinct Wnt-dependent developmental processes.     

Dapper 1 antagonizes Wnt signaling by promoting dishevelled degradation

Wnt signaling plays pivotal roles in the regulation of embryogenesis and cancer development. Xenopus Dapper (Dpr) was identified as an interacting protein for Dishevelled (Dvl), a Wnt signaling mediator, and modulates Wnt signaling. However, it is largely unclear how Dpr regulates Wnt signaling. Here, we present evidence that human Dpr1, the ortholog of Xenopus Dpr, inhibits Wnt signaling. We have identified the regions responsible for the Dpr-Dvl interaction in both proteins and found that the interaction interface is formed between the DEP (Dishevelled, Egl-10, and pleckstrin) domain of Dvl and the central and the C-terminal regions of Dpr1. The inhibitory function of human Dpr1 requires both its N and C terminus. Overexpression of the C-terminal region corresponding to the last 225 amino acids of Dpr1, in contrast to wild-type Dpr1, enhances Wnt signaling, suggesting a dominant negative function of this region. Furthermore, we have shown that Dpr1 induces Dvl degradation via a lysosome inhibitor-sensitive and proteasome inhibitor-insensitive mechanism. Knockdown of Dpr1 by RNA interference up-regulates endogenous Dvl2 protein. Taken together, our data indicate that the inhibitory activity of Dpr on Wnt signaling is conserved from Xenopus to human and that Dpr1 antagonizes Wnt signaling by inducing Dvl degradation.     

Circular RNA circEIF4G2 aggravates renal fibrosis in diabetic nephropathy by sponging miR‐218

Circular RNAs play essential roles in the development of various human diseases. However, how circRNAs are involved in diabetic nephropathy (DN) are not fully understood. Our study aimed to investigate the effects of circRNA circEIF4G2 on DN. Experiments were performed in the db/db mouse model of type 2 diabetes and NRK‐52E cells. We found that circEIF4G2 was significantly up‐regulated in the kidneys of db/db mice and NRK‐52E cells stimulated by high glucose. circEIF4G2 knockdown inhibited the expressions of TGF‐β1, Collagen I and Fibronectin in high glucose‐stimulated NRK‐52E cells, which could be rescued by miR‐218 inhibitor. Knockdown of SERBP1 reduced the expression of TGF‐β1, Collagen I and Fibronectin in HG‐stimulated NRK‐52E cells. In summary, our findings suggested that circEIF4G2 promotes renal tubular epithelial cell fibrosis via the miR‐218/SERBP1 pathway, presenting a novel insight for DN treatment.     

Dapper1 is a nucleocytoplasmic shuttling protein that negatively modulates Wnt signaling in the nucleus

Wnt signaling, via the activation of the canonical beta-catenin and lymphoid enhancer factor (LEF)/T-cell factor pathway, plays an important role in embryogenesis and cancer development by regulating the expression of genes involved in cell proliferation, differentiation, and survival. Dapper (Dpr), as a Dishevelled interactor, has been suggested to modulate Wnt signaling by promoting Dishevelled degradation. Here, we provide evidence that Dpr1 shuttles between the cytoplasm and the nucleus. Although overexpressed Dpr1 was mainly found in the cytoplasm, endogenous Dpr1 was localized over the cell, and Wnt1 induced its nuclear export. Treatment with leptomycin B induced nuclear accumulation of both endogenous and overexpressed Dpr1. We further identified the nuclear localization signal and the nuclear export signal within Dpr1. Using reporter assay and in vivo zebrafish embryo assay, we demonstrated that the forced nuclearly localized Dpr1 possessed the ability to antagonize Wnt signaling. Dpr1 interacted with beta-catenin and LEF1 and disrupted their complex formation. Furthermore, Dpr1 could associate with histone deacetylase 1 (HDAC1) and enhance the LEF1-HDAC1 interaction. Together, our findings suggest that Dpr1 negatively modulates the basal activity of Wnt/beta-catenin signaling in the nucleus by keeping LEF1 in the repressive state. Thus, Dpr1 controls Wnt/beta-catenin signaling in both the cytoplasm and the nucleus.     

Therapeutic Effects of Tangshen Formula on Diabetic Nephropathy in Rats

Objective

Inflammation and fibrosis are essential promoters in the pathogenesis of diabetic nephropathy (DN) in type 2 diabetes. The present study examined the anti-inflammation and anti-fibrosis effect of Tangshen Formula (TSF), a traditional Chinese medicine, on DN.

Research Design and Methods

Protective role of TSF in DN was examined in a rat model of type 2 DN that was established by high-fat diet-fed and low-dose-streptozotocin injection. TSF was suspended in 0.5% CMC-Na solution and delivered by oral gavage at a dosage of 1.67g/Kg body weight/day. The therapeutic effects and mechanisms of TSF on diabetic kidney injury were examined.

Results

We found that TSF treatment for 20 weeks attenuated DN by significantly inhibiting urinary excretion of albumin and renal histological injuries. These beneficial effects were associated with an inactivation of NF-κB signaling, thereby blocking the upregulation of pro-inflammatory cytokines (IL-1β, TNFα), chemokine (MCP-1), and macrophage infiltration in the TSF-treated rats with type 2 DN. In addition, TSF treatment also inactivated TGF-β/Smad3 signaling and therefore suppressed renal fibrosis including expressions of fibronectin, collagen I, and collagen IV. Further studies revealed that the inhibitory effect of TSF on TGF-β/Smad3 and NF-κB signaling in DN was associated with inhibition of Smurf2-dependent ubiquitin degradation of Smad7.

Conclusions

The present study reveals that TSF has therapeutic potential for type 2 DN in rats. Blockade of NF-κB-driven renal inflammation and TGF-β/Smad3-mediated renal fibrosis by preventing the Smurf2-mediated Smad7 degradation pathway may be mechanisms through which TSF inhibits type 2 DN.     

AMPK agonist alleviate renal tubulointerstitial fibrosis via activating mitophagy in high fat and streptozotocin induced diabetic mice

Renal tubulointerstitial fibrosis was a crucial pathological feature of diabetic nephropathy (DN), and renal tubular injury might associate with abnormal mitophagy. In this study, we investigated the effects and molecular mechanisms of AMPK agonist metformin on mitophagy and cellular injury in renal tubular cell under diabetic condition. The high fat diet (HFD) and streptozotocin (STZ)-induced type 2 diabetic mice model and HK-2 cells were used in this study. Metformin was administered in the drinking water (200 mg/kg/d) for 24 weeks. Renal tubulointerstitial lesions, oxidative stress and some indicators of mitophagy (e.g., LC3II, Pink1, and Parkin) were examined both in renal tissue and HK-2 cells. Additionally, compound C (an AMPK inhibitor) and Pink1 siRNA were applied to explore the molecular regulation mechanism of metformin on mitophagy. We found that the expression of p-AMPK, Pink1, Parkin, LC3II, and Atg5 in renal tissue of diabetic mice was decreased obviously. Metformin reduced the levels of serum creatinine, urine protein, and attenuated renal oxidative injury and fibrosis in HFD/STZ induced diabetic mice. In addition, Metformin reversed mitophagy dysfunction and the over-expression of NLRP3. In vitro pretreatment of HK-2 cells with AMPK inhibitor compound C or Pink1 siRNA negated the beneficial effects of metformin. Furthermore, we noted that metformin activated p-AMPK and promoted the translocation of Pink1 from the cytoplasm to mitochondria, then promoted the occurrence of mitophagy in HK-2 cells under HG/HFA ambience. Our results suggested for the first time that AMPK agonist metformin ameliorated renal oxidative stress and tubulointerstitial fibrosis in HFD/STZ-induced diabetic mice via activating mitophagy through a p-AMPK-Pink1-Parkin pathway.Subject terms: Diabetes complications, End-stage renal disease, Experimental models of disease     

Cyclin D1 is not an immediate target of beta-catenin following Apc loss in the intestine

Cyclin D1 is postulated to be a target of the canonical Wnt pathway and critical for intestinal adenoma development. We show here that, unlike cyclin D1 reporter assays, endogenous cyclin D1 levels are not affected following antagonism of the Wnt pathway in vitro, nor is cyclin D1 immediately up-regulated following conditional loss of Apc in vivo. Cyclin D1 levels do, however, increase in a delayed manner in a small subset of cells, suggesting such up-regulation occurs as a secondary event. We also analyzed the immediate consequences of Apc loss in a cyclin D1(-/-) background and failed to find any cyclin D1-dependent phenotypes. However, we did observe elevated cyclin D1 expression in lesions developing 20 days after Apc loss. In these circumstances, all adenomas (but not smaller lesions) showed cyclin D1 up-regulation. Finally in a smaller study, we analyzed whether cyclin D1 deficiency affected adenoma formation 20 days following induced loss of Apc. Unlike AhCre(+) Apc(fl/fl) mice (which all developed adenomas), doubly mutant AhCre(+) Apc(fl/fl) cyclin D1(-/-) mice only developed small lesions. Taken together, this argues that cyclin D1 up-regulation in intestinal neoplasia is important for tumor progression rather than initiation.