Jean S. Campbell

Design and synthesis of pH-responsive polymeric carriers that target uptake and enhance the intracellular delivery of oligonucleotides.

The delivery of biomolecular therapeutics that function intracellularly remains a significant challenge in the field of biotechnology. In this report, a new family of polymeric drug carriers that combine cell targeting, a pH-responsive membrane-disruptive component, and serum-stabilizing polyethylene glycol (PEG) grafts, is shown to direct the uptake and endosomal release of oligonucleotides in a primary hepatocyte cell line. These polymers are called encrypted polymers and are graft terpolymers that consist of a hydrophobic, membrane-disruptive backbone onto which hydrophilic PEG chains have been grafted through acid-degradable linker acetal linkages. In this report, the ability of the encrypted polymers to deliver rhodamine-labeled oligonucleotides or PEG-FITC (a model macromolecular drug) (5 kDa) into the cytoplasm of hepatocytes was investigated by fluorescence microscopy. Two new encrypted polymer derivatives (polymers E2 and E3) were synthesized that contained lactose for targeting to hepatocytes. Polymer E2 also has PEG-FITC conjugated to it, as a model macromolecular drug, and polymer E3 contains a pendant hexalysine moiety for complexing oligonucleotides. The results of the fluorescence microscopy experiments show that the encrypted polymers direct vesicular escape and efficiently deliver oligonucleotides and macromolecules into the cytoplasm of hepatocytes.

New Concepts in Liver Regeneration

The unique ability of the liver to regenerate itself has fascinated biologists for years and has made it the prototype for mammalian organ regeneration. Harnessing this process has great potential benefit in the treatment of liver failure and has been the focus of intense research over the past 50 years. Not only will detailed understanding of cell proliferation in response to injury be applicable to other dysfunction of organs, it may also shed light on how cancer develops in a cirrhotic liver, in which there is intense pressure on cells to regenerate. Advances in molecular techniques over the past few decades have led to the identification of many regulatory intermediates, and pushed us onto the verge of an explosive era in regenerative medicine. To date, more than 10 clinical trials have been reported in which augmented regeneration using progenitor cell therapy has been attempted in human patients. This review traces the path that has been taken over the last few decades in the study of liver regeneration, highlights new concepts in the field, and discusses the challenges that still stand between us and clinical therapy.

The mitogen-activated protein kinase pathway can mediate growth inhibition and proliferation in smooth muscle cells. Dependence on the availability of downstream targets.

Activation of the classical mitogen-activated protein kinase (MAPK) pathway leads to proliferation of many cell types. Accordingly, an inhibitor of MAPK kinase, PD 098059, inhibits PDGF-induced proliferation of human arterial smooth muscle cells (SMCs) that do not secrete growth-inhibitory PGs such as PGE2. In striking contrast, in SMCs that express the inducible form of cyclooxygenase (COX-2), activation of MAPK serves as a negative regulator of proliferation. In these cells, PDGF-induced MAPK activation leads to cytosolic phospholipase A2 activation, PGE2 release, and subsequent activation of the cAMP-dependent protein kinase (PKA), which acts as a strong inhibitor of SMC proliferation. Inhibition of either MAPK kinase signaling or of COX-2 in these cells releases them from the influence of the growth-inhibitory PGs and results in the subsequent cell cycle traverse and proliferation. Thus, the MAPK pathway mediates either proliferation or growth inhibition in human arterial SMCs depending on the availability of specific downstream enzyme targets.

Expression of suppressors of cytokine signaling during liver regeneration

The cytokines TNF and IL-6 play a critical role early in liver regeneration following partial hepatectomy (PH). Since IL-6 activates signal transducers and activators of transcription (STATs), we examined whether the suppressors of cytokine signaling (SOCS) may be involved in terminating IL-6 signaling. We show here that SOCS-3 mRNA is induced 40-fold 2 hours after surgery. SOCS-2 and CIS mRNA are only weakly induced, and SOCS-1 is not detectable. SOCS-3 induction after PH is transient and correlates with a decrease in STAT-3 DNA binding and a loss of tyrosine 705 phosphorylation. This response is markedly reduced in IL-6 knockout (KO) mice. TNF injection induces SOCS-3 mRNA in wild-type mice (albeit weakly compared with the increase observed after PH) but not in TNF receptor 1 or IL-6 KO mice. In contrast, IL-6 injection induces SOCS-3 in these animals, demonstrating a requirement for IL-6 in SOCS-3 induction. IL-6 injection into wild-type mice also induces SOCS-1, -2, and CIS mRNA, in addition to SOCS-3. Together, these results suggest that SOCS-3 may be a key component in downregulating STAT-3 signaling after PH and that SOCS-3 mRNA levels in the regenerating liver are regulated by IL-6.

The MAP kinase cascade.

Publisher Summary The discovery of a number of hormones and growth factors with receptors that are protein tyrosine kinases generated interest in discovering the pathway by which these signals are transmitted from these receptors to various parts of the cell. This chapter provides an overview of the two broad approaches that were employed to discover the pathway: (1) upstream approach and (2) downstream approach. The upstream approach shows that partially purified microtubule associated protein 2 (MAP-2) protein kinase obtained from insulin stimulates 3T3-L1 cells can phosphorylate and reactive a homogeneous phosphatase-treated S6 kinase. MAP-2 kinase, in essence a S6 kinase kinase, also appears to be regulated by serine/threonine phosphorylation because protein phosphatise 2A (PP2A) treatment inactivatsed the enzymatic activity. The chapter presents a downstream approach, which suggests that MAPKK could bind directly to p21 ras or indirectly through another protein and also MAPKK interacts with the Raf-1 C-terminal catalytic domain. These findings suggest that p21 ras and that MAPKK can form a complex with Raf-1 by binding to opposite ends of the protein. This chapter also discusses the features of MAP kinase cascade such as, inactivation of the cascade, functions and branch points, and cross talk with other kinases involved in signal transduction.

Transforming growth factor-beta differentially regulates oval cell and hepatocyte proliferation†

Oval cells are hepatocytic precursors that proliferate in late‐stage cirrhosis and that give rise to a subset of human hepatocellular carcinomas. Although liver regeneration typically occurs through replication of existing hepatocytes, oval cells proliferate only when hepatocyte proliferation is inhibited. Transforming growth factor‐β (TGF‐β) is a key inhibitory cytokine for hepatocytes, both in vitro and in vivo. Because TGF‐β levels are elevated in chronic liver injury when oval cells arise, we hypothesized that oval cells may be less responsive to the growth inhibitory effects of this cytokine. To examine TGF‐β signaling in vivo in oval cells, we analyzed livers of rats fed a choline‐deficient, ethionine‐supplemented (CDE) diet for phospho‐Smad2. Phospho‐Smad2 was detected in more than 80% of hepatocytes, but staining was substantially reduced in oval cells. Ki67 staining, in contrast, was significantly more common in oval cells than hepatocytes. To understand the inverse relationship between TGF‐β signaling and proliferation in oval cells and hepatocytes, we examined TGF‐β signaling in vitro. TGF‐β caused marked growth inhibition in primary hepatocytes and the AML12 hepatocyte cell line. Two oval cell lines, LE/2 and LE/6, were less responsive. The greater sensitivity of the hepatocytes to TGF‐β–induced growth inhibition may result from the absence of Smad6 in these cells. Conclusion: Our results indicate that oval cells, both in vivo and in vitro, are less sensitive to TGF‐β–induced growth inhibition than hepatocytes. These findings further suggest an underlying mechanism for the proliferation of oval cells in an environment inhibitory to hepatocytic proliferation. (HEPATOLOGY 2007;45:31–41.)

Regulation of liver regeneration and hepatocarcinogenesis by suppressor of cytokine signaling 3

Suppressor of cytokine signaling 3 (SOCS3) down-regulates several signaling pathways in multiple cell types, and previous data suggest that SOCS3 may shut off cytokine activation at the early stages of liver regeneration (Campbell, J.S., L. Prichard, F. Schaper, J. Schmitz, A. Stephenson-Famy, M.E. Rosenfeld, G.M. Argast, P.C. Heinrich, and N. Fausto. 2001.J. Clin. Invest. 107:1285–1292). We developed Socs3 hepatocyte-specific knockout (Socs3 h-KO) mice to directly study the role of SOCS3 during liver regeneration after a two-thirds partial hepatectomy (PH). Socs3 h-KO mice demonstrate marked enhancement of DNA replication and liver weight restoration after PH in comparison with littermate controls. Without SOCS3, signal transducer and activator of transcription 3 (STAT3) phosphorylation is prolonged, and activation of the mitogenic extracellular signal-regulated kinase 1/2 (ERK1/2) is enhanced after PH. In vitro, we show that SOCS3 deficiency enhances hepatocyte proliferation in association with enhanced STAT3 and ERK activation after epidermal growth factor or interleukin 6 stimulation. Microarray analyses show that SOCS3 modulates a distinct set of genes, which fall into diverse physiological categories, after PH. Using a model of chemical-induced carcinogenesis, we found that Socs3 h-KO mice develop hepatocellular carcinoma at an accelerated rate. By acting on cytokines and multiple proliferative pathways, SOCS3 modulates both physiological and neoplastic proliferative processes in the liver and may act as a tumor suppressor.

Disruption of redox homeostasis in tumor necrosis factor-induced apoptosis in a murine hepatocyte cell line.

Tumor necrosis factor (TNF) is a mediator of the acute phase response in the liver and can initiate proliferation and cause cell death in hepatocytes. We investigated the mechanisms by which TNF causes apoptosis in hepatocytes focusing on the role of oxidative stress, antioxidant defenses, and mitochondrial damage. The studies were conducted in cultured AML12 cells, a line of differentiated murine hepatocytes. As is the case for hepatocytes in vivo, AML12 cells were not sensitive to cell death by TNF alone, but died by apoptosis when exposed to TNF and a small dose of actinomycin D (Act D). Morphological signs of apoptosis were not detected until 6 hours after the treatment and by 18 hours approximately 50% of the cells had died. Exposure of the cells to TNF+Act D did not block NFkappaB nuclear translocation, DNA binding, or its overall transactivation capacity. Induction of apoptosis was characterized by oxidative stress indicated by the loss of NAD(P)H and glutathione followed by mitochondrial damage that included loss of mitochondrial membrane potential, inner membrane structural damage, and mitochondrial condensation. These changes coincided with cytochrome C release and the activation of caspases-8, -9, and -3. TNF-induced apoptosis was dependent on glutathione levels. In cells with decreased levels of glutathione, TNF by itself in the absence of transcriptional blocking acted as an apoptotic agent. Conversely, the antioxidant alpha-lipoic acid, that protected against the loss of glutathione in cells exposed to TNF+Act D completely prevented mitochondrial damage, caspase activation, cytochrome C release, and apoptosis. The results demonstrate that apoptosis induced by TNF+Act D in AML12 cells involves oxidative injury and mitochondrial damage. As injury was regulated to a larger extent by the glutathione content of the cells, we suggest that the combination of TNF+Act D causes apoptosis because Act D blocks the transcription of genes required for antioxidant defenses.

Reciprocal and Coordinate Regulation of Serum Amyloid A Versus Apolipoprotein A-I and Paraoxonase-1 by Inflammation in Murine Hepatocytes

Objectives—During inflammation, the serum amyloid A (SAA) content of HDL increases, whereas apolipoprotein A-I (apoA-I) and paraoxonase-1 (PON-1) decrease. It remains unclear whether SAA physically displaces apoA-I or if these changes derive from coordinated but inverse transcriptional regulation of the HDL apolipoprotein genes. Because cytokines stimulate the hepatic expression of inflammatory markers, we investigated their role in regulating SAA, apoA-I, and PON-1 expression. Methods and Results—A cytokine mixture (tumor necrosis factor [TNF]-&agr;, interleukin [IL]-1&bgr;, and IL-6) simultaneously induced SAA and repressed apoA-I and PON-1 expression levels. These effects were partially inhibited in cells pretreated with either nuclear factor &kgr;B (NF-&kgr;B) inhibitors (pyrrolidine dithiocarbamate, SN50, and overexpression of super-repressor inhibitor &kgr;B) or after exposure to the peroxisome proliferator-activated receptor-&agr; (PPAR&agr;) ligands (WY-14643 and fenofibrate). Consistent with these findings, the basal level of SAA was increased, whereas apoA-I and PON-1 decreased in primary hepatocytes from PPAR&agr;-deficient mice as compared with wild-type mice. Moreover, neither WY-14643 nor fenofibrate had any effect on SAA, apoA-I, or PON-1 expression in the absence of PPAR&agr;. Conclusion—These results suggest that cytokines increase the expression of SAA through NF-&kgr;B transactivation, while simultaneously decreasing the expression of apoA-I and PON-1 by inhibiting PPAR&agr; activation. Inflammation may convert HDL de novo into a more proatherogenic form by coordinate but inverse transcriptional regulation in the liver, rather than by physical displacement of apoA-I by SAA.

Proinflammatory Cytokine Production in Liver Regeneration Is Myd88-Dependent, but Independent of Cd14, Tlr2, and Tlr4

TNF and IL-6 are considered to be important to the initiation or priming phase of liver regeneration. However, the signaling pathways that lead to the production of these cytokines after partial hepatectomy (PH) have not been identified. Enteric-derived LPS appears to be important to liver regeneration, possibly by stimulating proinflammatory cytokine production after surgery. To determine whether LPS signaling pathways are involved in the regulation of the proinflammatory cytokines TNF and IL-6 during the priming phase of liver regeneration, we performed PH on mice lacking the TLRs Tlr4 and Tlr2, the LPS coreceptor, Cd14, and Myd88, an adapter protein involved in most TLR and IL-1R pathways. In MyD88 knockout (KO) mice after PH, both liver Tnf mRNA and circulating IL-6 levels were severely depressed compared with heterozygous or wild-type mice. Activation of STAT-3 and three STAT-3 responsive genes, Socs3, Cd14, and serum amyloid A2 were also blocked. In contrast, Tlr4, Tlr2, and Cd14 KO mice showed no deficits in the production of IL-6. Surprisingly, none of these KO mice showed any delay in hepatocyte replication. These data indicate that the LPS receptor TLR4, as well as TLR2 and CD14, do not play roles in regulating cytokine production or DNA replication after PH. In contrast, MyD88-dependent pathways appear to be responsible for TNF, IL-6, and their downstream signaling pathways.