Can Contrast-Enhanced Sonography Detect Bowel Wall Fibrosis in Mixed Inflammatory and Fibrotic Crohn Disease Lesions in an Animal Model?

OBJECTIVES: To determine whether contrast-enhanced sonographic quantitative perfusion parameters can detect bowel wall fibrosis in the setting of mixed inflammatory and fibrotic lesions in a Crohn disease animal model. METHODS: This study was approved by the institutional Committee on the Use and Care of Animals. Multiple (range, 1-5) 2,4,6-trinitrobenzenesulfonic acid-ethanol enemas were used to create intestinal inflammatory lesions with variable fibrosis in female Lewis rats. Low-mechanical index contrast-enhanced sonography was performed 3 days after the final enema using a 0.2-mL bolus of sulfur hexafluoride microbubbles injected through a tail vein. Contrast-enhanced sonographic data were analyzed with software that converts video data into echo-power (linearized) data. Colorectal lesions were scored for histopathologic inflammation and fibrosis; bowel wall collagen was quantified by Western blotting. The Spearman correlation was used to assess associations between contrast-enhanced sonographic quantitative parameters and bowel wall collagen; the Kruskal-Wallis test was used to compare continuous results between histopathologic groups. RESULTS: Thirty-one animals were included in our analysis. Animals were placed into 3 histopathologic cohorts: (1) severe bowel wall inflammation/minimal or no fibrosis (n = 11); (2) severe bowel wall inflammation/moderate fibrosis (n = 9); and (3) severe bowel wall inflammation/severe fibrosis (n = 11). Western blotting showed a significant difference in bowel wall collagen between histopathologic cohorts (P = .0001). There was no correlation between any contrast-enhanced sonographic quantitative parameter and bowel wall collagen (P > .05). There was no difference between histopathologic cohorts for any contrast-enhanced sonographic quantitative parameter (P > .05). CONCLUSIONS: Contrast-enhanced sonographic quantitative perfusion parameters failed to effectively detect bowel wall fibrosis in the setting of superimposed inflammation in a Crohn disease animal model.

Optimisation of Intestinal Fibrosis and Survival in the Mouse S. Typhimurium Model for Anti-fibrotic Drug Discovery and Preclinical Applications.

BACKGROUND AND AIMS: Intestinal fibrosis is a frequent complication in Crohn's disease [CD]. The mouse Salmonella typhimurium model, due to its simplicity, reproducibility, manipulability, and penetrance, is an established fibrosis model for drug discovery and preclinical trials. However, the severity of fibrosis and mortality are host- and bacterial strain-dependent, thus limiting the original model. We re-evaluated the S. typhimurium model to optimise fibrosis and survival, using commercially available mouse strains. METHODS: Fibrotic and inflammatory markers were evaluated across S. typhimurium ΔaroA:C57bl/6 studies performed in our laboratory. A model optimisation study was performed using three commercially available mouse strains [CBA/J, DBA/J, and 129S1/SvImJ] infected with either SL1344 or ΔaroA S. typhimurium. Fibrotic penetrance was determined by histopathology, gene expression, and αSMA protein expression. Fibrosis severity, penetrance, and survival were analysed across subsequent CBA studies. RESULTS: Fibrosis severity and survival are both host- and bacterial strain-dependent. Marked tissue fibrosis and 100% survival occurred in the CBA/J strain infected with SL1344. Subsequent experiments demonstrated that CBA/J mice develop extensive intestinal fibrosis, characterised by transmural tissue fibrosis, a Th1/Th17 cytokine response, and induction of pro-fibrotic genes and extracellular matrix proteins. A meta-analysis of subsequent SL1344:CBA/J studies demonstrated that intestinal fibrosis is consistent and highly penetrant across histological, protein, and gene expression markers. As proof-of-concept, we tested the utility of the SL1344:CBA/J fibrosis model to evaluate efficacy of CCG-203971, a novel anti-fibrotic drug. CONCLUSION: The S. typhimurium SL1344:CBA/J model is an optimised model for the study of intestinal fibrosis.

Characterizing intestinal inflammation and fibrosis in Crohn's disease by photoacoustic imaging: feasibility study.

The pathology of Crohn's disease (CD) is characterized by obstructing intestinal strictures because of inflammation (with high levels of hemoglobin), fibrosis (high levels of collagen), or a combination of both. The accurate characterization of the strictures is critical for the management of CD. This study examines the feasibility of characterizing intestinal strictures by Photoacoustic imaging (PAI) without extrapolation from superficial biopsies. Ex vivo normal rat colon tissue, inflammatory and fibrotic intestinal strictures in rat trinitrobenzene sulfonic acid (TNBS) model were first differentiated by a PA-US parallel imaging system. Surgically removed human intestinal stricture specimens were afterwards imaged by a multiwavelength acoustic resolution PA microscope (ARPAM). The experiment results suggest that PAI is a potential tool for the diagnosis of the diseased conditions in intestinal strictures.

Comparison of noncontrast MRI magnetization transfer and T2 -Weighted signal intensity ratios for detection of bowel wall fibrosis in a Crohn's disease animal model.

PURPOSE: To compare the abilities of magnetization transfer magnetic resonance imaging (MT-MRI) and T2 -weighted signal intensity (T2 WSI) ratios to detect intestinal fibrosis in a Crohn's disease animal model. MATERIALS AND METHODS: Ten rats ("Group 1") received one trinitrobenzenesulfonic acid enema to induce acute colonic inflammation, while 10 additional animals ("Group 2") received multiple enemas to induce colonic inflammation and fibrosis. Gradient recalled-echo MT-MRI (5 and 10 kHz off-resonance) and T2 -weighted spin-echo imaging were performed 2 days after the last enema. MT ratios (MTR) and T2 WSI ratios were calculated in the area of greatest colonic thickening. Bowel wall MTR, bowel wall MTR normalized to paraspinous muscle MTR ("normalized MTR"), and T2 WSI ratios were compared between animal groups using Student's t-test. RESULTS: At 10 kHz off-resonance, mean bowel wall MTR for Group 1 was 24.8 ± 3.1% vs. 30.3 ± 3.2% for Group 2 (P = 0.001). Mean normalized MTR was 0.45 ± 0.05 for Group 1 and 0.58 ± 0.08 for Group 2 (P = 0.0003). At 5 kHz off-resonance, mean bowel wall MTR for Group 1 was 34.7 ± 5.2% vs. 40.3 ± 3.6% for Group 2 (P = 0.015). Mean normalized MTR was 0.53 ± 0.08 for Group 1 and 0.64 ± 0.07 for Group 2 (P = 0.003). Mean T2 WSI ratio was 5.32 ± 0.98 for Group 1 and 3.01 ± 0.66 for group 2 (P < 0.0001). Mean T2 WSI ratio/MTR (10 kHz off-resonance) was 12.06 ± 2.70 for Group 1 and 5.22 ± 1.29 for Group 2 (P < 0.0001), with an ROC c-statistic of 0.98. CONCLUSION: MTR and T2 WSI ratios detect bowel wall fibrosis in a Crohn's disease animal model.

Ultrasound shear wave elastography helps discriminate low-grade from high-grade bowel wall fibrosis in ex vivo human intestinal specimens.

OBJECTIVES: To determine whether bowel wall fibrosis can be detected in freshly resected human intestinal specimens based on ultrasound-derived shear wave speed. METHODS: Seventeen intact (>3-cm) bowel segments (15 small and 2 large intestine) from 12 patients with known or suspected inflammatory bowel disease were procured immediately after surgical resection. Ultrasound shear wave elastography of the bowel wall was performed by two methods (Virtual Touch Quantification [VTQ] and Virtual Touch-IQ [VT-IQ]; Siemens Medical Solutions USA, Inc, Mountain View, CA). Eighteen short-axis shear wave speed measurements were acquired from each specimen: 3 from the 9-, 12-, and 3-o'clock locations for each method. Imaging was performed in two areas for specimens greater than 10 cm in length (separated by ≥5 cm). A gastrointestinal pathologist scored correlative histologic slides for inflammation and fibrosis. Differences in mean shear wave speed between bowel segments with low and high inflammation/fibrosis scores were assessed by a Student t test. Receiver operating characteristic curve analysis was performed. RESULTS: High-fibrosis score (n = 11) bowel segments had a significantly greater mean shear wave speed than low-fibrosis score (n = 6) bowel segments (mean ± SD: VTQ, 1.59 ± 0.37 versus 1.18 ± 0.08 m/s; P= .004; VT-IQ, 1.87 ± 0.44 versus 1.50 ± 0.26 m/s; P= .049). There was no significant difference in mean shear wave speed between high-and low-inflammation score bowel segments (P > .05 for both VTQ and VT-IQ). Receiver operating characteristic curves showed areas under the curve of 0.91 (95% confidence interval, 0.67-0.99) for VTQ and 0.77 (95% confidence interval, 0.51-0.94) for VT-IQ in distinguishing low-from high-fibrosis score bowel segments. CONCLUSIONS: Ex vivo bowel wall shear wave speed measurements increase when transmural intestinal fibrosis is present.

CD73 (ecto-5'-nucleotidase) hepatocyte levels differ across mouse strains and contribute to mallory-denk body formation.

UNLABELLED: Formation of hepatocyte Mallory-Denk bodies (MDBs), which are aggregates of keratins 8 and 18 (K8/K18), ubiquitin, and the ubiquitin-binding protein, p62, has a genetic predisposition component in humans and mice. We tested the hypothesis that metabolomic profiling of MDB-susceptible C57BL and MDB-resistant C3H mouse strains can illuminate MDB-associated pathways. Using both targeted and unbiased metabolomic analyses, we demonstrated significant differences in intermediates of purine metabolism. Further analysis revealed that C3H and C57BL livers differ significantly in messenger RNA (mRNA) level, protein expression, and enzymatic activity of the adenosine-generating enzyme, ecto-5'-nucleotidase (CD73), which was significantly lower in C57BL livers. CD73 mRNA levels were also dramatically decreased in human liver biopsies from hepatitis C and nonalcoholic fatty liver disease patients. Feeding mice with a diet containing the MDB-inducing agent, 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC), significantly decreased CD73 protein and activity in C57BL livers and resulted in loss of plasma membrane CD73 expression and activity in isolated mouse hepatocytes. To further examine the role of CD73 in MDB formation in vivo, we fed wild-type (WT) and CD73(-/-) mice a DDC-containing diet. Liver enlargement, p62 induction, and disappearance of the K8/K18 cytoskeleton were attenuated in CD73(-/-) , compared to WT livers. MDB formation, as assessed by biochemical and immunofluorescence detection of keratin and ubiquitin complexes, was nearly absent in CD73(-/-) mice. CONCLUSION: Purine metabolism and CD73 expression are linked to susceptibility to MDB formation in livers of different mouse strains. Expression of the adenosine-generating enzyme, CD73, contributes to experimental MDB induction and is highly regulated in MDB-associated liver injury in mice and in chronic human liver disease.

US elastography-derived shear wave velocity helps distinguish acutely inflamed from fibrotic bowel in a Crohn disease animal model.

PURPOSE: To determine if acoustic radiation force impulse elastography-derived bowel wall shear wave velocity (SWV) allows distinction of acutely inflamed from fibrotic intestine in a Crohn disease animal model. MATERIALS AND METHODS: University Committee on the Use and Care of Animals approval was obtained. An acute inflammation Crohn disease model was produced by treating eight Lewis rats with a single administration of trinitrobenzenesulfonic acid (TNBS) enema, with imaging performed 2 days later in the surviving six rats. Colonic fibrosis in an additional eight Lewis rats was achieved by administering repeated TNBS enemas during 4 weeks, with imaging performed in the surviving seven rats 7 days later to allow acute inflammation resolution. Nine transcutaneous bowel wall SWV measurements were obtained from the colon in all rats without and with applied strain. Mean SWVs without and with applied strain were compared between animal cohorts by using the Student t test, and receiver operating characteristic (ROC) curves were created to assess diagnostic performance. RESULTS: Mean bowel wall SWVs were significantly higher for fibrotic versus acute inflammation cohort of rats at 0% (3.4 ± 1.1 vs 2.3 ± 0.5 m/sec; P = .047) and 30% (6.3 ± 2.2 vs 3.6 ± 0.9 m/sec; P = .02) applied strain. Both acute inflammation and fibrotic cohort of rats demonstrated linear increases in mean SWV with increasing applied strain, with significantly different mean slopes (P = .02) and y-intercepts (P = .02). The area under the ROC curve of the SWV ratio (mean SWV/applied strain) for differentiating histopathologically confirmed fibrotic from inflamed bowel was 0.971. CONCLUSION: Bowel wall SWV helps distinguish acutely inflamed from fibrotic intestine in a Crohn disease animal model.

The hypoxia-inducible factor-C/EBPα axis controls ethanol-mediated hepcidin repression.

Hepcidin is a liver-derived peptide hormone and the master regulator of systemic iron homeostasis. Decreased hepcidin expression is a common feature in alcoholic liver disease (ALD) and in mouse models of ethanol loading. Dysregulation of hepcidin signaling in ALD leads to liver iron deposition, which is a major contributing factor to liver injury. The mechanism by which hepcidin is regulated following ethanol treatment is unclear. An increase in liver hypoxia was observed in an acute ethanol-induced liver injury model. The hypoxic response is controlled by a family of hypoxia-inducible transcription factors (HIFs), which are composed of an oxygen-regulated alpha subunit (HIFα) and a constitutively present beta subunit, aryl hydrocarbon receptor nuclear translocator (HIFß/Arnt). Disruption of liver HIF function reversed the repression of hepcidin following ethanol loading. Mouse models of liver HIF overexpression demonstrated that both HIF-1α and HIF-2α contribute to hepcidin repression in vivo. Ethanol treatment led to a decrease in CCAAT-enhancer-binding protein alpha (C/EBPα) protein expression in a HIF-dependent manner. Importantly, adenoviral rescue of C/EBPα in vivo ablated the hepcidin repression in response to ethanol treatment or HIF overexpression. These data provide novel insight into the regulation of hepcidin by hypoxia and indicate that targeting HIFs in the liver could be therapeutic in ALD.

Oxidative stress, Nrf2 and keratin up-regulation associate with Mallory-Denk body formation in mouse erythropoietic protoporphyria.

UNLABELLED: Mallory-Denk bodies (MDBs) are hepatocyte inclusions commonly seen in steatohepatitis. They are induced in mice by feeding 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) for 12 weeks, which also causes porphyrin accumulation. Erythropoietic protoporphyria (EPP) is caused by mutations in ferrochelatase (fch), and a fraction of EPP patients develop liver disease that is phenocopied in Fech(m1Pas) mutant (fch/fch) mice, which have an inactivating fch mutation. fch/fch mice develop spontaneous MDBs, but the molecular factors involved in their formation and whether they relate to DDC-induced MDBs are unknown. We tested the hypothesis that fch mutation creates a molecular milieu that mimics experimental drug-induced MDBs. In 13- and 20-week-old fch/fch mice, serum alkaline phosphatase, alanine aminotransferase, and bile acids were increased. The 13-week-old fch/fch mice did not develop histologically evident MDBs but manifested biochemical alterations required for MDB formation, including increased transglutaminase-2 and keratin overexpression, with a greater keratin 8 (K8)-to-keratin 18 (K18) ratio, which are critical for drug-induced MDB formation. In 20-week-old fch/fch mice, spontaneous MDBs were readily detected histologically and biochemically. Short-term (3-week) DDC feeding markedly induced MDB formation in 20-week-old fch/fch mice. Under basal conditions, old fch/fch mice had significant alterations in mitochondrial oxidative-stress markers, including increased protein oxidation, decreased proteasomal activity, reduced adenosine triphosphate content, and Nrf2 (redox sensitive transcription factor) up-regulation. Nrf2 knockdown in HepG2 cells down-regulated K8, but not K18. CONCLUSION: Fch/fch mice develop age-associated spontaneous MDBs, with a marked propensity for rapid MDB formation upon exposure to DDC, and therefore provide a genetic model for MDB formation. Inclusion formation in the fch/fch mice involves oxidative stress which, together with Nrf2-mediated increase in K8, promotes MDB formation.

Intestinal fibrosis is reduced by early elimination of inflammation in a mouse model of IBD: impact of a "Top-Down" approach to intestinal fibrosis in mice.

BACKGROUND: The natural history of Crohn's disease follows a path of progression from an inflammatory to a fibrostenosing disease, with most patients requiring surgical resection of fibrotic strictures. Potent antiinflammatory therapies reduce inflammation but do not appear to alter the natural history of intestinal fibrosis. The aim of this study was to determine the relationship between intestinal inflammation and fibrogenesis and the impact of a very early "top-down" interventional approach on fibrosis in vivo. METHODS: In this study we removed the inflammatory stimulus from the Salmonella typhimurium mouse model of intestinal fibrosis by eradicating the S. typhimurium infection with levofloxacin at sequential timepoints during the infection. We evaluated the effect of this elimination of the inflammatory stimulus on the natural history of inflammation and fibrosis as determined by gross pathology, histopathology, mRNA expression, and protein expression. RESULTS: Fibrogenesis is preceded by inflammation. Delayed eradication of the inflammatory stimulus by antibiotic treatment represses inflammation without preventing fibrosis. Early intervention significantly ameliorates but does not completely prevent subsequent fibrosis. CONCLUSIONS: This study demonstrates that intestinal fibrosis develops despite removal of an inflammatory stimulus and elimination of inflammation. Early intervention ameliorates but does not abolish subsequent fibrosis, suggesting that fibrosis, once initiated, is self-propagating, suggesting that a very early top-down interventional approach may have the most impact on fibrostenosing disease.

Ultrasound elasticity imaging for detecting intestinal fibrosis and inflammation in rats and humans with Crohn's disease.

BACKGROUND: Intestinal fibrosis causes many complications of Crohn's disease (CD). Available biomarkers and imaging modalities lack sufficient accuracy to distinguish intestinal inflammation from fibrosis. Transcutaneous ultrasound elasticity imaging (UEI) is a promising, noninvasive approach for measuring tissue mechanical properties. We hypothesized that UEI could differentiate inflammatory from fibrotic bowel wall changes in both animal models of colitis and humans with CD. METHODS: Female Lewis rats underwent weekly trinitrobenzene sulfonic acid enemas yielding models of acute inflammatory colitis (n = 5) and chronic intestinal fibrosis (n = 6). UEI scanning used a novel speckle-tracking algorithm to estimate tissue strain. Resected bowel segments were evaluated for evidence of inflammation and fibrosis. Seven consecutive patients with stenotic CD were studied with UEI and their resected stenotic and normal bowel segments were evaluated by ex vivo elastometry and histopathology. RESULTS: Transcutaneous UEI normalized strain was able to differentiate acutely inflamed (-2.07) versus chronic fibrotic (-1.10) colon in rat models of inflammatory bowel disease (IBD; P = .037). Transcutaneous UEI normalized strain also differentiated stenotic (-0.87) versus adjacent normal small bowel (-1.99) in human CD (P = .0008), and this measurement also correlated well with ex vivo elastometry (r = -0.81). CONCLUSIONS: UEI can differentiate inflammatory from fibrotic intestine in rat models of IBD and can differentiate between fibrotic and unaffected intestine in a pilot study in humans with CD. UEI represents a novel technology with potential to become a new objective measure of progression of intestinal fibrosis. Prospective clinical studies in CD are needed.

Fibrinogen-γ proteolysis and solubility dynamics during apoptotic mouse liver injury: heparin prevents and treats liver damage.

UNLABELLED: Fas ligand (FasL)-mediated hepatocyte apoptosis occurs in the context of acute liver injury that can be accompanied by intravascular coagulation (IC). We tested the hypothesis that analysis of selected protein fractions from livers undergoing apoptosis will shed light on mechanisms that are involved in liver injury that might be amenable to intervention. Proteomic analysis of the major insoluble liver proteins after FasL exposure for 4-5 hours identified fibrinogen-γ (FIB-γ) dimers and FIB-γ-containing high molecular mass complexes among the major insoluble proteins visible via Coomassie blue staining. Presence of the FIB-γ-containing products was confirmed using FIB-γ-specific antibodies. The FIB-γ-containing products partition selectively and quantitatively into the liver parenchyma after inducing apoptosis. Similar formation of FIB-γ products occurs after acetaminophen administration. The observed intrahepatic IC raised the possibility that heparin therapy may ameliorate FasL-mediated liver injury. Notably, heparin administration in mice 4 hours before or up to 2 hours after FasL injection resulted in a dramatic reduction of liver injury-including liver hemorrhage, serum alanine aminotransferase, caspase activation, and liver apoptosis-compared with heparin-untreated mice. Heparin did not directly interfere with FasL-induced apoptosis in isolated hepatocytes, and heparin-treated mice survived the FasL-induced liver injury longer compared with heparin-untreated animals. There was a sharp, near-simultaneous rise in FasL-induced intrahepatic apoptosis and coagulation, with IC remaining stable while apoptosis continued to increase. CONCLUSION: Formation of FIB-γ dimers and their high molecular mass products are readily detectable within the liver during mouse apoptotic liver injury. Heparin provides a potential therapeutic modality, because it not only prevents extensive FasL-related liver injury but also limits the extent of injury if given at early stages of injury exposure.

Rapamycin inhibits growth factor-induced cell cycle regulation in pancreatic beta cells.

A progressive decline in islet function is a major obstacle to success of islet transplantation. The cause of this decline is islet function is unclear, but immunosuppressive agents may contribute. Insulin-like growth factor-I (IGF-I) and betacellulin are important for islet cell survival and/or proliferation. In the present study, we performed studies of IGF-I and betacellulin on progression of islet cells through the cell cycle and the impact of immunosuppressive agents. Treatment of INS-1 cells for 24 hours with 20 ng/mL betacellulin or 50 ng/mL IGF-1 increased cells in S phase by ~2-fold. Treatment of INS-1 cells with IGF-I or betacellulin also increased cyclin D1 expression and nuclear exclusion of the cyclindependent kinase inhibitors p21(Cip1) and p27(Kip1). In INS-1 cells and islets, betacellulin- and IGF-I increased the increase in p70(s6 kinase) phosphorylation stimulated by betacellulin- and IGF-I in INS-1 cells. Rapamycin also inhibited betacellulin- and IGF-I IN IGF-1 cells. Rapamycin also inhibited betacellulin- and IGF-I-induced entry of cells into S phase and 5'-Bromo-2'-deoxyuridine incorporation as well as the effect of betacellulin and IGF-I on cyclin D1 expression and nuclear exclusion of p21(Cip1) and p(27Kip1). Together, these data suggest that the effect of betacellulin and IGF-I on islet cell growth and proliferation is mediated, in part, via signaling through mammalian target of rapamycin. As rapamycin is used to treat islet transplant recipients, these results suggest that rapamycin could have deleterious effects on islet proliferation and function over time.

Cdk4 is indispensable for postnatal proliferation of the anterior pituitary.

For proper development and tissue homeostasis, cell cycle progression is controlled by multilayered mechanisms. Recent studies using knock-out mice have shown that animals can develop relatively normally with deficiency for each of the G1/S-regulatory proteins, D-type and E-type cyclins, cyclin-dependent kinase 4 (Cdk4), and Cdk2. Although Cdk4-null mice show no embryonic lethality, they exhibit specific endocrine phenotypes, i.e. dwarfism, infertility, and diabetes. Here we have demonstrated that Cdk4 plays an essential non-redundant role in postnatal proliferation of the anterior pituitary. Pituitaries from wild-type and Cdk4-null embryos at embryonic day 17.5 are morphologically indistinguishable with similar numbers of cells expressing a proliferating marker, Ki67, and cells expressing a differentiation marker, growth hormone. In contrast, anterior pituitaries of Cdk4-null mice at postnatal 8 weeks are extremely hypoplastic with markedly decreased numbers of Ki67+ cells, suggesting impaired cell proliferation. Pituitary hyperplasia induced by transgenic expression of human growth hormone-releasing hormone (GHRH) is significantly diminished in the Cdk4+/- genetic background and completely abrogated in the Cdk4-/- background. Small interfering RNA (siRNA)-mediated knockdown of Cdk4 inhibits GHRH-induced proliferation of GH3 somato/lactotroph cells with restored expression of GHRH receptors. Cdk4 siRNA also inhibits estrogen-dependent cell proliferation in GH3 cells and closely related GH4 cells. In contrast, Cdk6 siRNA does not diminish proliferation of these cells. Furthermore, Cdk4 siRNA does not affect GHRH-induced proliferation of mouse embryonic fibroblasts or estrogen-dependent proliferation of mammary carcinoma MCF-7 cells. Taken together, Cdk4 is dispensable for prenatal development of the pituitary or proliferation of other non-endocrine tissues but indispensable specifically for postnatal proliferation of somato/lactotrophs.

The cyclin-dependent kinase inhibitors p27Kip1 and p21Cip1 cooperate to restrict proliferative life span in differentiating ovarian cells.

The timing of cellular exit from the cell cycle during differentiation is specific for each cell type or lineage. Granulosa cells in the ovary establish quiescence within several hours after the ovulation-inducing luteinizing hormone surge, whereas they undergo differentiation into corpora lutea. The expression of Cdk inhibitors p21(Cip1/Waf1) and p27(Kip1) is up-regulated during this process, suggesting that these cell cycle inhibitors are involved in restricting proliferative capacity of differentiating granulosa cells. Here we demonstrate that the lack of p27(Kip1) and p21(Cip1) synergistically renders granulosa cells extended an proliferative life span. Immunohistochemical analyses demonstrated that corpora lutea of p27(Kip1), p21(Cip1) double-null mice showed large numbers of cells with bromodeoxyuridine incorporation and high proliferative cell nuclear antigen expression, which were more remarkable than those in p27(Kip1) single-deficient mice showing modest hyperproliferation. In contrast, differentiating granulosa cells in p21(Cip1)-deficient mice ceased proliferation similarly to those in wild-type mice. Interestingly, granulosa cells isolated from p27(Kip1), p21(Cip1) double-null mice exhibited markedly prolonged proliferative life span in culture, unlike cells with other genotypes. Cultured p27(Kip1), p21(Cip1) double-null granulosa cells maintained expression of steroidogenic enzymes and gonadotropin receptors through 8-10 passages and could undergo further differentiation in responses to cAMP accumulation. Thus, the cooperation of p27(Kip1) and p21(Cip1) is critical for withdrawal of granulosa cells from the cell cycle, in concert with luteal differentiation and possibly culture-induced senescence.

Cdk4 deficiency inhibits skin tumor development but does not affect normal keratinocyte proliferation.

Most human tumors have mutations that result in deregulation of the cdk4/cyclin-Ink4-Rb pathway. Overexpression of D-type cyclins or cdk4 and inactivation of Ink4 inhibitors are common in human tumors. Conversely, lack of cyclin D1 expression results in significant reduction in mouse skin and mammary tumor development. However, complete elimination of tumor development was not observed in these models, suggesting that other cyclin/cdk complexes play an important role in tumorigenesis. Here we described the effects of cdk4 deficiency on mouse skin proliferation and tumor development. Cdk4 deficiency resulted in a 98% reduction in the number of tumors generated through the two-stage carcinogenesis model. The absence of cdk4 did not affect normal keratinocyte proliferation and both wild-type and cdk4 knockout epidermis are equally affected after topical treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), resulting in epidermal hyperplasia. In similar fashion, cdk4 knockout keratinocytes proliferated well in an in vivo model of wound-induced proliferation. Biochemical studies in mouse epidermis showed that cdk6 activity increased twofold in cdk4-deficient mice compared to wild-type siblings. These results suggest that therapeutic approaches to inhibit cdk4 activity could provide a target to inhibit tumor development with minimal or no effect in normal tissue.

Pituitary hypoplasia and lactotroph dysfunction in mice deficient for cyclin-dependent kinase-4.

The lactotroph undergoes dynamic regulation of cell cycle progression during pregnancy, as well as throughout the development of the pituitary. We recently reported that female mice with targeted disruption of Cdk4, one of the G(1)-regulatory cyclin-dependent kinases, are unable to support embryo implantation because of defective progesterone secretion from the corpus luteum. In this study, we demonstrate that this phenotype is not attributable to a primary defect in the corpus luteum but is a consequence of defective prolactin (PRL) production caused by inappropriate development of the pituitary lactotroph population. Specifically, the pituitary of Cdk4-deficient mice is extremely hypoplastic. Lactotrophs and somatotrophs of prepubertal Cdk4-deficient mice were 80% decreased in number, relative to those in wild-type mice, whereas gonadotrophs were unaffected. Lactotrophs of Cdk4-deficient mice did not proliferate in response to estrogen administration, whereas estrogen could induce the expression of galanin, an estrogen-responsive factor required for lactotroph proliferation. The reduction in lactotroph numbers was reflected by markedly diminished serum PRL levels in both prepubertal and postcoital Cdk4-deficient mice. Administration of PRL, after mating, significantly increased serum progesterone levels and restored implantation in Cdk4-deficient female mice. These observations demonstrate that Cdk4 is required for normal proliferation of the lactotroph population.

Intact follicular maturation and defective luteal function in mice deficient for cyclin- dependent kinase-4.

Cell cycle progression of granulosa cells is critical for ovarian function, especially follicular maturation. During follicular maturation, FSH induces cyclin D2, which promotes G1 progression by activating cyclin-dependent kinase-4 (Cdk4). Because cyclin D2-deficient mice exhibit a block in follicular growth, cyclin D2/Cdk4 has been hypothesized to be required for FSH-dependent proliferation of granulosa cells. Here we investigate ovarian function in Cdk4-knockout mice we recently generated. Cdk4(-/-) females were sterile, but the morphology of their ovaries appeared normal before sexual maturation. The number of preovulatory follicles and the ovulation efficiency were modestly reduced in gonadotropin-treated Cdk4(-/-) mice. However, unlike cyclin D2-deficient mice, Cdk4(-/-) mice showed no obvious defect in FSH-induced proliferation of granulosa cells. Cdk4(-/-) ovaries displayed normal preovulatory expression of aromatase, PR, and cyclooxygenase-2. Postovulatory progesterone secretion was markedly impaired in Cdk4(-/-) mice, although granulosa cells initiated luteinization with induction of p450 side-chain cleavage cytochrome and p27(Kip1). Progesterone treatment rescued implantation and restored fertility in Cdk4(-/-) mice. Serum PRL levels after mating were significantly reduced in Cdk4(-/-) mice, suggesting the involvement of perturbed PRL regulation in luteal failure. Thus, Cdk4 is critical for luteal function, and some redundant protein(s) can compensate for the absence of Cdk4 in proliferation of granulosa cells.