David A. Rudnick

Protein N-myristoylation.

Protein N-myristoylation refers to the covalent attachment of myristic acid, a 14-carbon saturated fatty acid (C14:0), to the N-terminal glycine of proteins. Linkage occurs via an amide bond and takes place as proteins are being synthesized. N-myristoylproteins have varied intracellular destinations, and are involved in myriad cellular functions ranging from signal transduction to protein and vesicular trafficking. N-myristoylproteins are encountered in members of all kingdoms of the eukaryotic domain (Protist, Fungi, Plant, and Animal), but are not produced by members of Bacteria or Archaea. MyristoylCoA: protein N-myristoyltransferase (Nmt), E.C. 2.3.1.97, a member of the GCN5 acetyltransferase (GNAT) superfamily, is responsible for catalyzing the transfer of myristate from myristoylCoA to proteins. While the acylCoA substrate specificity of Nmt has been highly conserved during evolution, its peptide substrate specificities have diverged among eukaryotes.

Disruption of hepatic adipogenesis is associated with impaired liver regeneration in mice

The liver responds to injury with regulated tissue regeneration. During early regeneration, the liver accumulates fat. Neither the mechanisms responsible for nor the functional significance of this transient steatosis have been determined. In this study, we examined patterns of gene expression associated with hepatic fat accumulation in regenerating liver and tested the hypothesis that disruption of hepatic fat accumulation would be associated with impaired hepatic regeneration. First, microarray‐based gene expression analysis revealed that several genes typically induced during adipocyte differentiation were specifically upregulated in the regenerating liver prior to peak hepatocellular fat accumulation. These observations suggest that hepatic fat accumulation is specifically regulated during liver regeneration. Next, 2 methods were employed to disrupt hepatocellular fat accumulation in the regenerating liver. Because exogenous leptin supplementation reverses hepatic steatosis in leptin‐deficient mice, the effects of leptin supplementation on liver regeneration in wild‐type mice were examined. The data showed that leptin supplementation resulted in suppression of hepatocellular fat accumulation and impairment of hepatocellular proliferation during liver regeneration. Second, because glucocorticoids regulate cellular fat accumulation during adipocyte differentiation, the effects of hepatocyte‐specific disruption of the glucocorticoid receptor were similarly evaluated. The results showed that hepatic fat accumulation and hepatocellular proliferation were also suppressed in mice with liver specific disruption of glucocorticoid receptor. In conclusion, suppression of hepatocellular fat accumulation is associated with impaired hepatocellular proliferation following partial hepatectomy, indicating that hepatocellular fat accumulation is specifically regulated during and may be essential for normal liver regeneration. (HEPATOLOGY 2004;40:1322–1332.)

Alpha-1-antitrypsin deficiency: A new paradigm for hepatocellular carcinoma in genetic liver disease †

Liver disease in alpha‐1‐antitrypsin (α1AT) deficiency is caused by a gain‐of‐toxic function mechanism engendered by the accumulation of a mutant glycoprotein in the endoplasmic reticulum (ER). The extraordinary degree of variation in phenotypical expression of this liver disease is believed to be determined by genetic modifiers and/or environmental factors that influence the intracellular disposal of the mutant glycoprotein or the signal transduction pathways that are activated. Recent investigations suggest that a specific repertoire of signaling pathways are involved, including the autophagic response, mitochondrial‐ and ER‐caspase activation, and nuclear factor kappaB (NFκB) activation. Whether activation of these signaling pathways, presumably to protect the cell, inadvertently contributes to liver injury or perhaps protects the cell from one injury and, in so doing, predisposes it to another type of injury, such as hepatocarcinogenesis, is not yet known. Recent studies also suggest that hepatocytes with marked accumulation of α1ATZ, globule‐containing hepatocytes, engender a cancer‐prone state by surviving with intrinsic damage and by chronically stimulating in ‘trans’ adjacent relatively undamaged hepatocytes that have a selective proliferative advantage. Further, this paradigm may apply to other genetic and infectious liver diseases that are predisposed to hepatocellular carcinoma. (HEPATOLOGY 2005.)

Prostaglandins are required for CREB activation and cellular proliferation during liver regeneration.

The liver responds to multiple types of injury with an extraordinarily well orchestrated and tightly regulated form of regeneration. The response to partial hepatectomy has been used as a model system to elucidate the molecular basis of this regenerative response. In this study, we used cyclooxygenase (COX)-selective antagonists and -null mice to determine the role of prostaglandin signaling in the response of liver to partial hepatectomy. The results show that liver regeneration is markedly impaired when both COX-1 and COX-2 are inhibited by indocin or by a combination of the COX-1 selective antagonist, SC-560, and the COX-2 selective antagonist, SC-236. Inhibition of COX-2 alone partially inhibits regeneration whereas inhibition of COX-1 alone tends to delay regeneration. Neither the rise in IL-6 nor the activation of signal transducer and activator of transcription-3 (STAT3) that is seen during liver regeneration is inhibited by indocin or the selective COX antagonists. In contrast, indocin treatment prevents the activation of CREB by phosphorylation that occurs during hepatic regeneration. These data indicate that prostaglandin signaling is required during liver regeneration, that COX-2 plays a particularly important role but COX-1 is also involved, and implicate the activation of CREB rather than STAT3 as the mediator of prostaglandin signaling during liver regeneration.

Analyses of hepatocellular proliferation in a mouse model of α‐1‐antitrypsin deficiency

α‐1‐Antitrypsin (α1‐AT) deficiency is the most common cause of metabolic pediatric liver disease. Hepatocellular injury is caused by toxicity of the mutant α‐1‐antitrypsin Z (α1‐ATZ) molecule retained within hepatocytes. In these studies, we used the PiZ transgenic mouse model of α1‐AT deficiency to examine hepatocellular proliferation in response to chronic liver injury resulting from this metabolic disease. The results showed increased hepatocellular proliferation and caspase 9 activation in male PiZ mice compared with female PiZ and wild‐type mice. Hepatic α1‐AT mRNA and protein expression also were increased in male PiZ mice, suggesting that greater hepatocellular proliferation and caspase activation in males results from increased hepatotoxicity associated with greater intracellular α1‐ATZ accumulation. Testosterone treatment of female PiZ mice increased α1‐ATZ expression and hepatocellular proliferation to a level comparable with that in males. In PiZ mice, hepatocytes devoid of intracellular α1‐AT globules had a proliferative advantage compared with globule‐containing hepatocytes. However, this advantage is relative because both globule‐containing and globule‐devoid hepatocytes exhibited comparable proliferation after partial hepatectomy. In conclusion, these data indicate that intracellular retention of mutant α1‐ATZ is associated with a regenerative stimulus leading to increased hepatocellular proliferation, that gender‐specific signals influence the degree of α1‐AT expression and associated hepatic injury, and that hepatocytes devoid of α1‐ATZ have a proliferative advantage over cells that accumulate the mutant protein. This selective proliferation suggests that hepatocellular transplantation may be applicable for treatment of this and other slowly progressive metabolic liver diseases (HEPATOLOGY 2004;39:1048–1055.)

Delayed hepatocellular mitotic progression and impaired liver regeneration in early growth response-1-deficient mice.

The early growth response-1 transcription factor (Egr-1) is induced as part of the immediate-early gene expression response during early liver regeneration. In the studies reported here the functional significance of EGR-1 expression during liver regeneration was examined by characterizing the hepatic regenerative response to partial hepatectomy in Egr-1 null mice. The results of these studies showed that liver regeneration in Egr-1 null mice is impaired. Although activation of interleukin-6-STAT3 signaling, regulation of expression of hepatic C/ebpα, C/ebpβ, cyclin D, and cyclin E and progression through the first wave of hepatocellular DNA synthesis occurred appropriately following partial hepatectomy in Egr-1 null mice, subsequent signaling events and cell cycle progression after the first round of DNA synthesis were deranged. This derangement was characterized by increased activation of the p38 mitogen-activated protein kinase and inhibition of hepatocellular metaphase-to-anaphase mitotic progression. Together these observations suggest that EGR-1 is an important regulator of hepatocellular mitotic progression. In support of this, microarray-based gene expression analysis showed that induction of expression of the cell division cycle 20 gene (Cdc20), a key regulator of the mitotic anaphase-promoting complex, is significantly reduced in Egr-1 null mice. Taken together these data define a novel functional role for EGR-1 in regulating hepatocellular mitotic progression through the spindle assembly checkpoint during liver regeneration.

Intravenous N-acetylcysteine in pediatric patients with nonacetaminophen acute liver failure: A placebo-controlled clinical trial

N‐acetylcysteine (NAC) was found to improve transplantation‐free survival in only those adults with nonacetaminophen (non‐APAP) acute liver failure (ALF) and grade 1‐2 hepatic encephalopathy (HE). Because non‐APAP ALF differs significantly between children and adults, the Pediatric Acute Liver Failure (PALF) Study Group evaluated NAC in non‐APAP PALF. Children from birth through age 17 years with non‐APAP ALF enrolled in the PALF registry were eligible to enter an adaptively allocated, doubly masked, placebo‐controlled trial using a continuous intravenous infusion of NAC (150 mg/kg/day in 5% dextrose in water [D5W]) or placebo (D5W) for up to 7 days. The primary outcome was 1‐year survival. Secondary outcomes included liver transplantation‐free survival, liver transplantation (LTx), length of intensive care unit (ICU) and hospital stays, organ system failure, and maximum HE score. A total of 184 participants were enrolled in the trial with 92 in each arm. The 1‐year survival did not differ significantly (P = 0.19) between the NAC (73%) and placebo (82%) treatment groups. The 1‐year LTx‐free survival was significantly lower (P = 0.03) in those who received NAC (35%) than those who received placebo (53%), particularly, but not significantly so, among those less than 2 years old with HE grade 0‐1 (NAC 25%; placebo 60%; P = 0.0493). There were no significant differences between treatment arms for hospital or ICU length of stay, organ systems failing, or highest recorded grade of HE. Conclusion: NAC did not improve 1‐year survival in non‐APAP PALF. One‐year LTx‐free survival was significantly lower with NAC, particularly among those <2 years old. These results do not support broad use of NAC in non‐APAP PALF and emphasizes the importance of conducting controlled pediatric drug trials, regardless of results in adults. (HEPATOLOGY 2013)

Rapamycin reduces intrahepatic alpha-1-antitrypsin mutant Z protein polymers and liver injury in a mouse model

Alpha-1-antitrypsin (a1AT) deficiency is caused by homozygosity for the a1AT mutant Z gene and occurs in one in 2000 Americans. The Z mutation confers an abnormal conformation on the a1AT mutant Z protein, resulting in accumulation within the endoplasmic reticulum of hepatocytes and chronic liver injury. Autophagy is one of several proteolytic mechanisms activated to cope with this hepatocellular protein burden, and is likely important in disposal of the unique polymerized conformation of the a1AT mutant Z protein, which is thought to be especially injurious to the cell. Recent data indicate that rapamycin may more efficiently upregulate autophagy when given in weekly dose pulses, as compared with a daily regimen. Therefore, we evaluated the effect of rapamycin on PiZ mice, a well-characterized model which recapitulates human a1AT liver disease. Daily dosing had no effect on autophagy, on accumulation of a1AT mutant Z protein or on liver injury. Weekly dosing of rapamycin did increase autophagic activity, as shown by increased numbers of autophagic vacuoles. This was associated with reduction in the intrahepatic accumulation of a1AT mutant Z protein in the polymerized conformation. Markers of hepatocellular injury, including cleavage of caspase 12 and hepatic fibrosis, were also decreased. In conclusion, this is the first report of a successful in vivo method for reduction of intrahepatic a1AT mutant Z polymerized protein. Application of this finding may be therapeutic in patients with a1AT deficiency by reducing the intracellular burden of the polymerized, mutant Z protein and by reducing the progression of liver injury.

Directed microtubule growth, +TIPs, and kinesin-2 are required for uniform microtubule polarity in dendrites.

BACKGROUND in many differentiated cells, microtubules are organized into polarized noncentrosomal arrays, yet few mechanisms that control these arrays have been identified. For example, mechanisms that maintain microtubule polarity in the face of constant remodeling by dynamic instability are not known. Drosophila neurons contain uniform-polarity minus-end-out microtubules in dendrites, which are often highly branched. Because undirected microtubule growth through dendrite branch points jeopardizes uniform microtubule polarity, we have used this system to understand how cells can maintain dynamic arrays of polarized microtubules. RESULTS we find that growing microtubules navigate dendrite branch points by turning the same way, toward the cell body, 98% of the time and that growing microtubules track along stable microtubules toward their plus ends. Using RNAi and genetic approaches, we show that kinesin-2, and the +TIPS EB1 and APC, are required for uniform dendrite microtubule polarity. Moreover, the protein-protein interactions and localization of Apc2-GFP and Apc-RFP to branch points suggests that these proteins work together at dendrite branches. The functional importance of this polarity mechanism is demonstrated by the failure of neurons with reduced kinesin-2 to regenerate an axon from a dendrite. CONCLUSIONS we conclude that microtubule growth is directed at dendrite branch points and that kinesin-2, APC, and EB1 are likely to play a role in this process. We propose that kinesin-2 is recruited to growing microtubules by +TIPS and that the motor protein steers growing microtubules at branch points. This represents a newly discovered mechanism for maintaining polarized arrays of microtubules.

p21 is Required for Dextrose-Mediated Inhibition of Mouse Liver Regeneration

The inhibitory effect of dextrose supplementation on liver regeneration was first described more than 4 decades ago. Nevertheless, the molecular mechanisms responsible for this observation have not been elucidated. We investigated these mechanisms using the partial hepatectomy model in mice given standard or 10% dextrose (D10)‐supplemented drinking water. The results showed that D10‐treated mice exhibited significantly reduced hepatic regeneration compared with controls, as assessed by hepatocellular bromodeoxyuridine (BrdU) incorporation and mitotic frequency. D10 supplementation did not suppress activation of hepatocyte growth factor (HGF), induction of transforming growth factor alpha (TGF‐α) expression, or tumor necrosis factor alpha–interleukin‐6 cytokine signaling, p42/44 extracellular signal‐regulated kinase (ERK) activation, immediate early gene expression, or expression of CCAAT/enhancer binding protein beta (C/EBPβ), but did augment expression of the mito‐inhibitory factors C/EBPα, p21Waf1/Cip1, and p27Kip1. In addition, forkhead box M1 (FoxM1) expression, which is required for normal liver regeneration, was suppressed by D10 treatment. Finally, D10 did not suppress either FoxM1 expression or hepatocellular proliferation in p21 null mice subjected to partial hepatectomy, establishing the functional significance of these events in mediating the effects of D10 on liver regeneration. Conclusion: These data show that the inhibitory effect of dextrose supplementation on liver regeneration is associated with increased expression of C/EBPα, p21, and p27, and decreased expression of FoxM1, and that D10‐mediated inhibition of liver regeneration is abrogated in p21‐deficient animals. Our observations are consistent with a model in which hepatic sufficiency is defined by homeostasis between the energy‐generating capacity of the liver and the energy demands of the body mass, with liver regeneration initiated when the functional liver mass is no longer sufficient to meet such demand. (HEPATOLOGY 2009.)