Alida Maria Koorts

Ferritin and ferritin isoforms I: Structure–function relationships, synthesis, degradation and secretion

Abstract Ferritin is the intracellular protein responsible for the sequestration, storage and release of iron. Ferritin can accumulate up to 4500 iron atoms as a ferrihydrite mineral in a protein shell and releases these iron atoms when there is an increase in the cells need for bioavailable iron. The ferritin protein shell consists of 24 protein subunits of two types, the H-subunit and the L-subunit. These ferritin subunits perform different functions in the mineralization process of iron. The ferritin protein shell can exist as various combinations of these two subunit types, giving rise to heteropolymers or isoferritins. Isoferritins are functionally distinct and characteristic populations of isoferritins are found depending on the type of cell, the proliferation status of the cell and the presence of disease. The synthesis of ferritin is regulated both transcriptionally and translationally. Translation of ferritin subunit mRNA is increased or decreased, depending on the labile iron pool and is controlled by an iron-responsive element present in the 5′-untranslated region of the ferritin subunit mRNA. The transcription of the genes for the ferritin subunits is controlled by hormones and cytokines, which can result in a change in the pool of translatable mRNA. The levels of intracellular ferritin are determined by the balance between synthesis and degradation. Degradation of ferritin in the cytosol results in complete release of iron, while degradation in secondary lysosomes results in the formation of haemosiderin and protection against iron toxicity. The majority of ferritin is found in the cytosol. However, ferritin with slightly different properties can also be found in organelles such as nuclei and mitochondria. Most of the ferritin produced intracellularly is harnessed for the regulation of iron bioavailability; however, some of the ferritin is secreted and internalized by other cells. In addition to the regulation of iron bioavailability ferritin may contribute to the control of myelopoiesis and immunological responses.

Ferritin and ferritin isoforms II: protection against uncontrolled cellular proliferation, oxidative damage and inflammatory processes.

Abstract Ferritin is a major iron storage protein involved in the regulation of iron availability. Each ferritin molecule comprises 24 subunits. Various combinations of H-subunits and L-subunits make up the 24-subunit protein structure and these ferritin isoforms differ in their H-subunit to L-subunit ratio, as well as in their metabolic properties. Ferritin is an acute-phase protein and its expression is up-regulated in conditions such as uncontrolled cellular proliferation, in any condition marked by excessive production of toxic oxygen radicals, and by infectious and inflammatory processes. Under such conditions ferritin up-regulation is predominantly stimulated by increased reactive oxygen radical production and by cytokines. The major function of ferritin in these conditions is to reduce the bio-availability of iron in order to stem uncontrolled cellular proliferation and excessive production of reactive oxygen radicals. Ferritin is not, however, indiscriminately up-regulated in these conditions as a marked shift towards a predominance in H-subunit rich ferritins occurs. Preliminary indications are that, while the L-subunit primarily fulfils the conventional iron storage role, the H-subunit functions primarily as rapid regulator of iron availability, and perhaps indirectly as regulator of other cellular processes. It is suggested that the optimum differential expression of the two subunits differ for different cells and under different conditions and that the expression of appropriate isoferritins offers protection against uncontrolled cellular proliferation, oxidative stress and against side effects of infectious and inflammatory conditions.

Pro- and Anti-Inflammatory Cytokines during Immune Stimulation: Modulation of Iron Status and Red Blood Cell Profile

Forty-eight patients were subdivided according to C-reactive protein (CRP) levels, resulting in 19 patients with normal (2.8 ± 2.8 mg/L) and 29 with elevated (82.2 ± 76.2 mg/L) CRP levels. The elevated CRP group had iron and red blood cell (RBC) profiles characteristic of chronic immune stimulation (CIS), and the normal CRP group, profiles of true iron deficiency. Normal relationships between storage iron, bioavailable iron, and RBC indices were absent in the elevated CRP group—implying the role of iron as major determinant of the RBC profile to be diminished during CIS. The elevated CRP group had significant increases in proinflammatory cytokines (INF-γ, TNF-α, Il-1β, Il-6, and Il-8). Anti-inflammatory cytokine levels were normal, except for Il-10, supporting previous indications that Il-10 contributes to reducing bioavailable iron. Regression analysis suggested decreases in transferrin to be related to increases in Il-8 and an increase in ferritin to be related to a decrease in Il-12 levels. TGF-β levels were positively related to transferrin and negatively to ferritin.

Acute Phase Proteins: Ferritin and Ferritin Isoforms

Ferritin is a positive acute phase reactant, exhibiting increased levels in blood during the acute phase response. Nevertheless, the precise role of ferritin as an acute phase reactant remains to be clarified. As for other acute phase proteins, ferritin is produced and secreted by hepatocytes. However, ferritin is also produced and secreted by other cell types, including macrophages and cancer cells. Many isoforms of ferritin (isoferritins) are found in the body, depending on the H-subunit to L-subunit ratio in the ferritin protein shell. The subunit composition of ferritin molecules is a major determinant of the functional properties of the ferritin isoforms. Expression of ferritin and its subunits is governed by the amount of metabolically available iron, the presence of oxidative stress and both proand antiinflammatory cytokines. Ferritin as an acute phase reactant is well known for its intracellular iron sequestration and storage abilities during immune activation (Weiss & Goodnough, 2005). This function is of high importance for protection of the body against microbial proliferation, oxidative damage, inflammation and cancer. Although the regulation of iron appears to be a primary function of ferritin in both normal conditions and during the acute phase response, other functions, beyond the control of iron bio-availability, have also been described. Although more investigations are required in order to clarify the precise role of ferritin as an acute phase reactant, this chapter shows a synopsis on the present knowledge on ferritin during the acute phase response. In the first part of this chapter (2-4), the processes of iron sequestration, storage and release by the ferritin molecule, the significance of the presence of isoferritins, and the regulation of the expression of ferritin by iron are described. In the next section (5) changes with diseases, and possible significance of extracellular (plasma) ferritin is discussed. In the last section (6-11) the differential up-regulation of H-subunit rich ferritins during the acute phase response, the importance of H-subunit rich ferritins in the withholding of iron by the macrophage, as well as its role in immune modulation, its pro-apoptotic and anti-apoptotic activities, and variations in cancer are addressed.

Intracellular free calcium in the neutrophils of maintenance haemodialysis patients

Chronic renal failure has on occasion been referred to as a state of calcium toxicity. The aim of this study was to investigate the status of intracellular free Ca2+ in the neutrophils of chronic renal failure patients on maintenance haemodialysis treatment. Factors previously suggested to influence intracellular free Ca2+ were investigated including PTH levels, oxidative stress and recombinant human erythropoietin administration. The study involved 14 chronic renal failure patients on the haemodialysis programme of the Pretoria Academic hospital. Intracellular free Ca2+ and transmembrane Ca2+ fluxes were investigated by fluorescence spectrophotometry. Increases above control values were found in intracellular free Ca2+ (P‐value 0·0242) and in the transmembrane Ca2+ flux upon fMLP stimulation (P‐value 0·0002). The results showed significant differences in intracellular free Ca2+ between patients on rHuEPO and patients not on rHuEPO. The apparently rHuEPO‐induced increase in intracellular free Ca2+ persisted in the presence of calcium channel blockers. No overt indications of oxidative stress could be detected by the antioxidant vitamin levels. It is concluded that factors other than those associated with uraemia, such as rHuEPO administration, might contribute to the often reported increase in intracellular free Ca2+ in these patients. Further studies to investigate the relationship between intracellular free Ca2+, rHuEPO and calcium channel blockers are suggested.

Expression of the H- and L-subunits of ferritin in bone marrow macrophages of patients with osteoarthritis

Osteoarthritis is a disease characterized by an increase in the production of reactive oxygen species (ROS) in afflicted joints. Excess iron, due to its role in the production of ROS and crystal deposition in the joints, is implicated in the disease progression of osteoarthritis. Ferritin is a major regulator of the bioavailability of iron, and its functions are determined largely by the combination of H- and L-subunits present in its outer protein shell. The purpose of the study was to investigate the expression of the H- and L-subunits of ferritin in bone marrow macrophages of osteoarthritis patients. The cytokine profiles were assessed as cytokines play an important role in the expression of the ferritin subunits. The H-subunit of ferritin in the bone marrow macrophages was significantly higher (P value = 0.035) in the osteoarthritis patients compared with the controls (107.84; 69.25–167.94 counts/μm2; n= 7 versus 71.07; 58.56–86.26 counts/μm2; n= 19). A marginally significant increase (P value = 0.059) was shown for the expression of the L-subunit in the osteoarthritis patients compared with the controls (133.03; 104.04–170.10 counts/μm2; n= 7 versus 104.23; 91.53–118.70 counts/μm2; n= 19). The osteoarthritis and control groups had comparable C-reactive protein, as well as proinflammatory and anti-inflammatory cytokine concentrations. The major exception was for transforming growth factor-β (TGF-β), which was higher (P value = 0.014) in the plasma of the osteoarthritis patients (16.69; 13.09–21.28 ng/mL; n= 7 versus 8.60; 6.34–11.67 ng/mL; n= 19). Up-regulation of the ferritin subunits decreases the levels of bioavailable iron and provides protection against the unwarranted production of ROS and crystal deposition. A role for TGF-β in the up-regulation of the expression of the H-subunit, and possibly the L-subunit, of ferritin is postulated in osteoarthritis.

Expression of the H-subunit and L-subunit of ferritin in bone marrow macrophages and cells of the erythron during cellular immune activation

BACKGROUND The expression of the two types of ferritin subunits, the H-subunit and L-subunit, has been shown to be differentially regulated by cytokines. The primary aim of the present study was to quantitatively measure the expression of the H-subunit and L-subunit of ferritin in bone marrow macrophages and cells of the erythron in patients with chronic T-helper cell type-1 immune stimulation. METHODS The expression of the H-subunit and L-subunit of ferritin in bone marrow macrophages and cells of the erythron was quantitatively evaluated by post-embedding immunolocalisation with immunogold transmission electron microscopy. RESULTS The present study showed up-regulation of the H-subunit of ferritin in the bone marrow macrophage in patients with pronounced cellular immune activation (94.7±37.3 counts/μm(2); n=31 vs 72.4±34.0 counts/μm(2); n=13, p-value=0.037). CONCLUSION This supports a possible role for H-subunit rich ferritins in the hypoferraemia of chronic disease.

Short-term fatty acid effects on adipocyte glucose uptake: mechanistic insights.

The modulation of insulin sensitivity in visceral fat tissue could be important in the treatment of Type 2 diabetes mellitus. Selected fatty acids may impact on insulin-stimulated and basal glucose uptake in adipocytes, thus isolated rat epididymal adipocytes were exposed to 100 microM oleic, arachidonic, eicosapentaenoic, docosahexaenoic or stearic acids and insulin (15 nM) or vehicle for 30 min. Glucose uptake was quantified by measuring uptake of 3H-deoxyglucose/mg adipocyte protein/min. Where appropriate, inhibitors were included to elucidate the mechanisms involved. In this model, insulin stimulated glucose uptake with 62+/-7%. All fatty acids tested, except for stearic acid, depressed insulin-stimulated glucose uptake by an average of 33+/-4.2%. On the other hand, all fatty acids tested except stearic and arachidonic acids, stimulated basal glucose uptake with an average of 34+/-8.1%. Inhibitor studies showed the involvement of prostaglandins, lipoxins, protein kinase C and tyrosine kinase in these processes.