Cyclin E1 controls proliferation of hepatic stellate cells and is essential for liver fibrogenesis in mice

Liver fibrogenesis is associated with the transition of quiescent hepatocytes and hepatic stellate cells (HSCs) into the cell cycle. Exit from quiescence is controlled by E-type cyclins (cyclin E1 [CcnE1] and cyclin E2 [CcnE2]). Thus, the aim of the current study was to investigate the contribution of E-type cyclins for liver fibrosis in man and mice. Expression of CcnE1, but not of its homolog, CcnE2, was induced in fibrotic and cirrhotic livers from human patients with different etiologies and in murine wild-type (WT) livers after periodical administration of the profibrotic toxin, CCl(4) . To further evaluate the potential function of E-type cyclins for liver fibrogenesis, we repetitively treated constitutive CcnE1(-/-) and CcnE2(-/-) knock-out mice with CCl(4) to induce liver fibrosis. Interestingly, CcnE1(-/-) mice were protected against CCl(4) -mediated liver fibrogenesis, as evidenced by reduced collagen type I α1 expression and the lack of septum formation. In contrast, CcnE2(-/-) mice showed accelerated fibrogenesis after CCl(4) treatment. We isolated primary HSCs from WT, CcnE1(-/-) , and CcnE2(-/-) mice and analyzed their activation, proliferation, and survival in vitro. CcnE1 expression in WT HSCs was maximal when they started to proliferate, but decreased after the cells transdifferentiated into myofibroblasts. CcnE1(-/-) HSCs showed dramatically impaired survival, cell-cycle arrest, and strongly reduced expression of alpha smooth muscle actin, indicating deficient HSC activation. In contrast, CcnE2-deficient HSCs expressed an elevated level of CcnE1 and showed enhanced cell-cycle activity and proliferation, compared to WT cells. Conclusions: CcnE1 and CcnE2 have antagonistic roles in liver fibrosis. CcnE1 is indispensable for the activation, proliferation, and survival of HSCs and thus promotes the synthesis of extracellular matrix and liver fibrogenesis. (HEPATOLOGY 2012;56:1140-1149).     

Impaired expression of acyl-CoA synthetase 5 in sporadic colorectal adenocarcinomas

Several pathways of fatty acid metabolism have been shown to be associated with the pathogenesis of colorectal cancer. Fatty acid acyl-CoA thioesters are formed from free fatty acids and coenzyme A by the activity of acyl-CoA synthetases (ACSs). Whilst an increase in ACS4 expression has been associated with colorectal carcinogenesis, little is known about possible pathogenetic functions of other ACS isoforms, such as ACS5, in tumourigenesis. In the present study, gene expression, protein synthesis, and enzymatic activity of ACS5 in sporadic colorectal adenocarcinomas, adenomas, and established cell lines were analysed using RT-PCR, western blot analysis, immunofluorescence, and an enzymatic assay. Enhanced expression of ACS5 mRNA and protein as well as enzymatic activity was found in adenomas and in 11 (73%; group 1) of 15 colorectal adenocarcinomas investigated, while a decrease of ACS5 was seen in four tumours (27%; group 2). However, basal ACS5 enzymatic activity was increased as a percentage of the total activity of ACSs in both groups, arguing for an absolute (group 1) or relative (group 2) increase in ACS5 enzymatic activity in all adenocarcinomas investigated. These findings are reflected by in vitro analysis of three established colorectal adenocarcinoma cell lines, in which activity of ACS5 occurred. The results suggest the involvement of ACS5 in the early genesis of colorectal cancer, most likely by modification of the transport and pool formation of long-chain acyl-CoA thioesters, as recently demonstrated for other isoforms of the ACS family.     

Sekundäres malignes Melanom der Gallenblase

The history of gallbladder involvement by a malignant melanoma in a 65-year-old woman is reported. The gallbladder, clinically resected for cholecystitis, harboured a polypoid dark pigmented tumour. The tumour was identified as a malignant melanoma immunohistochemically by positive reactions for gp100 (HMB45), melan A, and MiTF. Clinically, the patient was treated for cutaneous malignant melanoma by local excision 10 years earlier. The literature of pigmented lesions of the gallbladder is reviewed. In conclusion, the most important differential diagnosis of pigmented lesions of the gallbladder is the secondary gallbladder melanoma.     

A neuroprotective function for the hematopoietic protein granulocyte-macrophage colony stimulating factor (GM-CSF).

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic cytokine responsible for the proliferation, differentiation, and maturation of cells of the myeloid lineage, which was cloned more than 20 years ago. Here we uncovered a novel function of GM-CSF in the central nervous system (CNS). We identified the GM-CSF alpha-receptor as an upregulated gene in a screen for ischemia-induced genes in the cortex. This receptor is broadly expressed on neurons throughout the brain together with its ligand and induced by ischemic insults. In primary cortical neurons and human neuroblastoma cells, GM-CSF counteracts programmed cell death and induces BCL-2 and BCL-Xl expression in a dose- and time-dependent manner. Of the signaling pathways studied, GM-CSF most prominently induced the PI3K-Akt pathway, and inhibition of Akt strongly decreased antiapoptotic activity. Intravenously given GM-CSF passes the blood-brain barrier, and decreases infarct damage in two different experimental stroke models (middle cerebral artery occlusion (MCAO), and combined common carotid/distal MCA occlusion) concomitant with induction of BCL-Xl expression. Thus, GM-CSF acts as a neuroprotective protein in the CNS. This finding is remarkably reminiscent of the recently discovered functionality of two other hematopoietic factors, erythropoietin and granulocyte colony-stimulating factor in the CNS. The identification of a third hematopoietic factor acting as a neurotrophic factor in the CNS suggests a common principle in the functional evolution of these factors. Clinically, GM-CSF now broadens the repertoire of hematopoietic factors available as novel drug candidates for stroke and neurodegenerative diseases.     

The hematopoietic factor G-CSF is a neuronal ligand that counteracts programmed cell death and drives neurogenesis

G-CSF is a potent hematopoietic factor that enhances survival and drives differentiation of myeloid lineage cells, resulting in the generation of neutrophilic granulocytes. Here, we show that G-CSF passes the intact blood-brain barrier and reduces infarct volume in 2 different rat models of acute stroke. G-CSF displays strong anti-apoptotic activity in mature neurons and activates multiple cell survival pathways. Both G-CSF and its receptor are widely expressed by neurons in the CNS, and their expression is induced by ischemia, which suggests an autocrine protective signaling mechanism. Surprisingly, the G-CSF receptor was also expressed by adult neural stem cells, and G-CSF induced neuronal differentiation in vitro. G-CSF markedly improved long-term behavioral outcome after cortical ischemia, while stimulating neural progenitor response in vivo, providing a link to functional recovery. Thus, G-CSF is an endogenous ligand in the CNS that has a dual activity beneficial both in counteracting acute neuronal degeneration and contributing to long-term plasticity after cerebral ischemia. We therefore propose G-CSF as a potential new drug for stroke and neurodegenerative diseases.     

Regulation of enterocyte apoptosis by acyl-CoA synthetase 5 splicing

BACKGROUND AND AIMS: The constant renewal of enterocytes along the crypt-villus axis (CVA) of human small intestine is due to cell-inherent changes resulting in the apoptotic cell death of senescent enterocytes. The aim of the present study was to examine underlying molecular mechanisms of the cell death at the villus tip. METHODS: Characterization of human acyl-coenzyme A (CoA) synthetase 5 (ACSL5) was performed by cloning, recombinant protein expression, biochemical approaches, and several functional and in situ analyses. RESULTS: Our data show that different amounts of acyl-CoA synthetase 5-full length (ACSL5-fl) and a so far unknown splice variant lacking exon 20 (ACSL5-Delta 20) are found in human enterocytes. In contrast with the splice variant ACSL5-Delta 20, recombinant and purified ACSL5-fl protein is active at a highly alkaline pH. Over expression of ACSL5-fl protein is associated with a decrease of the anti-apoptotic FLIP protein in a ceramide-dependent manner and an increased cell-surface expression of the death receptor TRAIL-R1. Expression analyses revealed that the ACSL5-fl/ACSL5-Delta 20 ratio increases along the CVA, thereby sensitizing ACSL5-fl-dominated cells at the villus tip to the death ligand TRAIL, which is corroborated by functional studies with human small intestinal mucosal samples and an immortalized human small intestinal cell line. CONCLUSIONS: Our results suggest an ACSL5-dependent regulatory mechanism that contributes to the cellular renewal along the CVA in human small intestine. Deregulation of the ACSL5-fl/ACSL5-Delta 20 homeostasis in the maturation and shedding of cells along the CVA might also be of relevance for the development of intestinal neoplasia.     

Conversion of CO2 into organic acids by engineered autotrophic yeast

The increase of CO₂ emissions due to human activity is one of the preeminent reasons for the present climate crisis. In addition, considering the increasing demand for renewable resources, the upcycling of CO₂ as a feedstock gains an extensive importance to establish CO₂-neutral or CO₂-negative industrial processes independent of agricultural resources. Here we assess whether synthetic autotrophic Komagataella phaffii (Pichia pastoris) can be used as a platform for value-added chemicals using CO₂ as a feedstock by integrating the heterologous genes for lactic and itaconic acid synthesis. ¹³C labeling experiments proved that the resulting strains are able to produce organic acids via the assimilation of CO₂ as a sole carbon source. Further engineering attempts to prevent the lactic acid consumption increased the titers to 600 mg L⁻¹, while balancing the expression of key genes and modifying screening conditions led to 2 g L⁻¹ itaconic acid. Bioreactor cultivations suggest that a fine-tuning on CO₂ uptake and oxygen demand of the cells is essential to reach a higher productivity. We believe that through further metabolic and process engineering, the resulting engineered strain can become a promising host for the production of value-added bulk chemicals by microbial assimilation of CO₂, to support sustainability of industrial bioprocesses.

Between 2011 and 2020 the annual average CO₂ emission due to human activity exceeded 38 gigatons, of which around 22 gigatons are removed again from the atmosphere to terrestrial and ocean CO₂ sinks. This imbalance, mostly due to combustion of fossil fuels, causes the steady increase in CO₂ levels in the atmosphere which is one of the primary reasons for the climate crisis our planet is facing today (1).Biologically produced fuels and commodity chemicals bear the potential to counteract this deleterious development, but the most common feedstocks used for bioproduction, such as glucose, sucrose and starch rely on agricultural production and are bearing the risk to threaten food security. Using autotrophic microorganisms as production platforms exploits the potential of CO₂ itself as an alternative carbon source. In nature, autotrophic microorganisms play a major role in CO₂ fixation by fixing 200 gigatons of CO₂ every year (2). However, the rates of most natural microbial CO₂ fixing pathways are low: the photosynthetic efficiency in cyanobacteria is limited to 1 to 2% which makes industrial processes using photoautotrophs economically less feasible (3). Chemoautotrophs may overcome this barrier as their energy harvesting processes are more efficient than light harvesting of photoautotrophs.One well-known autotroph that is being developed for biological production of materials is Cupriavidus necator (formerly known as Ralstonia eutropha). By harvesting energy with a controlled Knallgas reaction these bacteria assimilate CO₂ via the Calvin-Benson-Bessham (CBB) cycle. Besides naturally produced polyhydroxyalkanoates (PHA), metabolic pathway engineering enabled the production of several other chemicals (4–7).Several chemolithotrophic bacteria were demonstrated as production hosts for various chemicals such as ethanol, 2,3-butanediol, butanol, isopropanol, acetone, or isobutyric acid via natural carbon assimilation pathways (8–11). In addition to natural CO₂ fixing microorganisms, implementation of heterologous CO₂ fixing pathways to heterotrophic microorganisms like Myceliophthora thermophila provided a mixotrophic strain that is able to produce malic acid with a higher yield compared to the parent strain in which only the reductive tricarboxylic acid (TCA) cycle is used for the production (12).Natural chemoautotrophs have a large potential to convert CO₂ to chemicals. However, they are often recalcitrant to genetic editing, have complex nutrient demands, or may require complex process technological solutions like the transfer of gaseous substrates and energy sources. To circumvent some of these limitations well established prokaryotic and eukaryotic production hosts have been engineered to assimilate CO₂. The bacterial workhorse Escherichia coli and the yeast Komagataella phaffii (Pichia pastoris) were provided with the CBB cycle, enabling them to assimilate CO₂ by using formate or methanol, respectively, as energy sources. Both engineered microorganisms can grow sustainably with CO₂ as carbon source (13, 14). Conceptually formate and methanol are regarded as sustainable feedstocks for biotechnology when they are derived from CO₂ by hydrogenation or electrochemical reduction (15).To make an impact on the global CO₂ household such autotrophic processes need to convert CO₂ into bulk products. Besides ethanol, short chain organic acids are the second largest group of chemicals manufactured by industrial biotechnology. The market of biologically produced organic acids is expected to reach more than $36 billion by 2026 (16). The annual bioproduction of some of the key organic acids (citric, acetic, lactic, succinic acid) is more than 12 million metric tons (17–20). Recently, itaconic acid has also gained attention as a promising chemical building block with an estimated market increase to 170 kilotons per year and $260 million in 2025 (21). Lactic and itaconic acid are feedstocks for polymer production so that they, and other biobased commodity chemicals compete for the annual polymer production of more than 300 million tons, a volume that denotes a significant impact on the global CO₂ balance.Here, we set out to evaluate if the autotrophic K. phaffii strain can be used as a platform for organic acid production. Synthetic autotrophy was introduced to K. phaffii by converting the native peroxisomal methanol assimilation pathway, the xylulose monophosphate (XuMP) cycle, into the CBB cycle (13). To achieve that, the formaldehyde assimilating enzyme dihydroxyacetone synthase (DAS) was replaced by a bacterial ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), and phosphoribulose kinase (PRK) from spinach was added to supply ribulose bisphosphate from XuMP precursors. Four yeast glycolytic enzymes were targeted to peroxisomes to close the CBB cycle to glyceraldehyde 3-phosphate (G3P), both as an intermediate and the product of the assimilation cycle (Fig. 1 A and B). In the present work, using modular synthetic biology tools, we implemented the genes for lactic and itaconic acid synthesis plus accessory genes into the K. phaffii genome and demonstrate that the autotrophic K. phaffii strain is capable of producing organic acids solely from CO₂ as carbon source.Open in a separate windowFig. 1.Expression of cadA and ldhL enables organic acid production in synthetic autotrophic K. phaffii. (A–D) Schematic pathways. (A) In wild-type K. phaffii methanol is oxidized to formaldehyde (black arrow) and assimilated in the XuMP cycle (orange arrows) or dissimilated to CO₂, respectively (purple arrow). (B) synthetic autotrophy in K. phaffii: the native assimilatory branch of methanol utilization was interrupted by deleting DAS1 and DAS2 (dashed gray line). AOX1 was knocked out to reduce the rate of formaldehyde formation which could be toxic to the cells. RuBisCO and PRK were integrated to complete a functional CBB cycle (green arrows). Additionally, two bacterial chaperones, groEL and groES, were overexpressed to assist the folding of RuBisCO. TDH3, PGK1, TKL1, TPI1 carrying each a peroxisomal targeting signal were overexpressed to assure the localization of the entire CBB cycle in peroxisomes. More details about the engineering strategy can be found in ref. (13). (C) Itaconic acid (red) and (D) lactic acid production (blue), (E) growth profiles, and (F) organic acid production profiles of the producing strains and the control. Time axis corresponds to the production phase under autotrophic conditions. At least three biological replicates were used in the screening to monitor the producing strains. Shades represent the SDs (±). 3PG: 3-phosphoglycerate, AcCoA: acetyl-coenzyme A, AOX1 and AOX2: alcohol oxidase 1 and 2, cadA: cis-aconitate decarboxylase, CBB cycle: Calvin-Benson-Bassham cycle, CISA_c: cytosolic cis-aconitate, CISA_m: mitochondrial cis-aconitate, DAS1 and DAS2: dihydroxyacetone synthase 1 and 2, DHA: dihydroxyacetone, FAL: formaldehyde, G3P: glyceraldehyde 3-phosphate, ITA: itaconic acid, LA: lactic acid, ldhL: L-lactate dehydrogenase, MeOH: methanol, mttA: mitochondrial tricarboxylic acid transporter, NAD⁺/NADH: nicotinamide adenine dinucleotide, PRK: phosphoribulokinase, PYR: pyruvate, RuBP: ribulose 1,5-bisphosphate, RuBisCO: ribulose 1,5-bisphosphate carboxylase/oxygenase, Xu5P: xylulose 5-phosphate, XuMP cycle: xylulose monophosphate cycle.