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REVIEW OF LITERATURE

1. ANEMIA

Anemia is a major public health problem affecting 1.62 billion people globally. Although
the prevalence of anemia in highly developed countries is 9% and in low developed
countries the prevalence is 43%. Children and women of reproductive age are most at
risk, with global anemia prevalence estimates of 47% in children younger than 5 years,
42% in pregnant women, and 30% in non-pregnant women aged 15–49 years, and with
Africa and Asia accounting for more than 85% of the absolute anemia burden in high risk
groups (McLean et al., 2009). Anemia is estimated to contribute to more than 115 000
maternal deaths and 591 000 perinatal deaths globally per year (Ezzati M.et al., 2004).
The consequences of morbidity associated with chronic anemia extend to loss of
productivity from impaired work capacity, cognitive impairment, and increased
susceptibility to infection, (Haas and Brownlie, 2001) which also exerts a substantial
economic burden.

Anemia is defined as the decrease in hemoglobin concentration, red cell count or packed
cell volume and the subsequent impairment in meeting the oxygen demand of tissues
(Warrell et al., 2003). The criteria adopted by WHO to define anemia status by
hemoglobin concentration are shown below Table 1 (WHO, 2008).

Table 1- Thresholds of haemoglobin used to define anemia in different sub-populations,

at sea level

Age groups Haemoglobin conc. (g/l)

Non-pregnant woman <15 years <120
Pregnant woman <110
Children (0.5-4.5 years) <110
Children (5-11.9 years) <115
Children (12-14.9 years) <120
Men >15 years <130

1.1 Determinants of anemia

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Determinants of the prevalence and distribution of anemia in a population involve a

complex interplay of political, ecological, social, and biological factors (Figure 1).

Figure 1: Conceptual model of the determinants of anemia

1.2 Causes and type of anemia

Classification

Anemia can be broadly classified into decreased erythrocyte production (ineffective

erythropoiesis) as a result of impaired proliferation of red-cell precursors or ineffective
maturation of erythrocytes; or increased loss of erythrocytes through increased
destruction (haemolysis) or blood loss; or both. These processes are broadly determined
by nutrition, infectious disease, and genetics (Tolentino and Friedman, 2007) (Figure 2).

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Fig 2: Causes of anemia in countries with low or middle incomes

Nutritional anemia

Nutritional anemia is caused by insufficient bioavailability of haemopoietic nutrients

needed to meet the requirements of hemoglobin and erythrocyte synthesis (Semba and
Bloem, 2008). As human diets have shifted over time from hunter-gatherer to more
cultivated cereal-based diets with more heat exposure during food preparation, there has
been a large drop in bioavailable haemopoietic nutrients (iron, vitamin B12 and folic
acid) and absorption enhancers such as vitamin C. This situation is compounded by
increased intake of other dietary factors that reduce the bioavailability of non-heme iron,
such as polyphenols (e.g. tea, coffee, and spices such as cinnamon), phytates (whole
grains, legumes), and calcium (dairy products) (Zimmermann et al., 2007).

 Iron deficiency anemia

Iron deficiency occurs when the intake of total or bioavailable iron is inadequate to meet
iron demands, or to compensate for increased losses. Iron has an important role in the
function of several biological processes, and is an integral part of the haemoglobin
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molecule wherein Fe²+ is bound to the protein proto porphyrin IX complex to form haem.
Lack of available iron thereby results in low heme concentrations and hypochromic
microcytic anemia.

 Folic acid deficiency

Folic acid is required for the synthesis and maturation of erythrocytes, and low serum and
erythrocyte concentrations of folate can lead to changes in cell morphology and
intramedullary death of erythrocytes and reduced erythrocyte lifespan. Folic acid
deficiency contributes to megaloblastic anemia, a condition characterized by cells with
large and malformed nuclei resulting from impaired DNA synthesis. During pregnancy,
folate demands increase, and women entering pregnancy with poor folate status often
develop megaloblastic anemia.

 Vitamin B12 deficiency

Vitamin B12 is synthesized only by microorganisms, and its primary source is from
ingestion of animal products. Absorption of vitamin B 12 involves a complex process by
which gastric enzymes and acid facilitate its release from food sources, before being
bound by an intrinsic factor secreted by gastric parietal cells, followed by uptake in the
distal ileum. Vitamin B12 deficiency can result in a megaloblastic macrocytic anemia,
which is more common in severe vitamin B12 deficiency (Metz, 2008).

 Vitamin A deficiency: Vitamin A plays an important part in erythropoiesis and has been
shown to improve haemoglobin concentration and increase the efficacy of iron
supplementation (Fishman SM et al., 2000). The mechanisms are not fully understood,
but are suggested to operate through effects on transferrin receptors affecting the
mobilisation of iron stores, increasing iron absorption, stimulating erythroid precursors in
the bone marrow, and reducing susceptibility to infections.
 Soil-transmitted helminths: Infectious diseases can contribute to anemia through
impaired absorption and metabolism of iron and other micronutrients or increased
nutrient losses. Of soil-transmitted helminth infections, hookworms (Necator americanus
and Ancylostoma duodenale) are the major cause of anemia, and are commonly found in
sub-Saharan Africa and south-east Asia, with an estimated 576–740 million infections
(Bethony J et al., 2006).

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 Malaria: Plasmodium falciparum is the most pathogenic species and can lead to severe
anemia, and subsequent hypoxia and congestive heart failure (Menendez et al., 2000).
The mechanisms of malaria-related anemia has evolved substantially, and can be broadly
characterised as increased erythrocyte destruction and decreased erythrocyte production,
with mechanisms probably acting simultaneously and affected by factors such as age,
pregnancy, malarial species, previous exposure and prophylaxis.
 Schistosomiasis: Postulated mechanisms include iron deficiency from blood loss from
haematuria (S. haematobium) and bloody diarrhoea (S. japonicum and S. mansoni);
splenic sequestration or increased haemolysis by the hypertrophic spleen, or both; auto
immune haemolysis; and anemia of chronic disease driven by pro-inflammatory cytokines
(Friedman, et al., 2005).
 HIV/AIDS: Anemia is the most common haematological complication associated with
HIV infection, and is a marker of disease progression and survival (Volberding et al.,
2004). The mechanism of HIV/AIDS-related anemia is multifactorial, resulting from HIV
infection and the induced anemia of chronic disease, AIDS-related illnesses, and
antiretroviral treatment.
 Genetic causes: Genetic haemoglobin disorders, which result from structural variation or
reduced production of globin chains of haemoglobin, can result in anemia. Estimates of
the burden of haemoglobin disorders suggest that at least 5·2% of the global population,
and more than 7% of pregnant women, are carriers of a significant haemoglobin variant
(Modell and Darlison, 2008).

2. Iron metabolism in humans

2.1 Storage of iron: The total amount of iron in a 70 kg man is about 3500–4000 mg,
corresponding to an average concentration of 50–60 mg of iron per kg of body weight.
The vast majority (2300 mg, 65%) of iron in the body is found in the haemoglobin of
erythrocytes. About a tenth of total iron (350 mg) is present in the myoglobin of muscle
and enzymes and cytochrome of other tissues. Of the remainder approximately 500 mg is
found in macrophages of the reticuloendothelial system, about 200-1000mg is stored in
hepatocytes in the form of ferritin while 150mg of iron is found in the bone marrow
(Munoz et al., 2011).

2.2 Absorption of iron

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Role of intestine in iron metabolism- An average western world diet includes ingestion of
about 15-20mg of iron, 10% of which it is present in heme form and the remainder in the
non-haem form. Most of the ingested heme iron is found in haemoglobin and myoglobin
of meat protein. The low gastric pH releases these proteins from the meat and subsequent
protease action in the stomach and intestine releases free heme. It has been proposed that
enterocyte absorbed haem via the Heme Carrier Protein1 transporter localized on their
brush border membrane. Non-haem iron is found in meat and plant foods and largely
exists in the ferric (Fe3+) form. Ferric iron, unlike ferrous (Fe2+) iron, is highly insoluble
and not easily absorbed. The low pH created in the stomach, as well as the presence of
dietary ascorbic acid reduce Fe3+ to Fe2+ ion enhancing solubility and absorption. The
duodenal cytochrome b (Dcytb) protein is localised on the brush-border membrane of the
enterocytes. Dcytb is a ferrireductase enzyme responsible for the reduction of Fe 3+ to Fe2+
ions. Following this reduction process, divalent Fe 2+ ions are transported into duodenal
enterocyte via divalent metal transporter 1(DMT1).DMT1 is brush border protein which
transports several divalent ion across the membrane.

When there is a low demand for iron in the body, absorbed iron is stored within the
enterocyte in the form of ferritin, an intracellular iron storage protein. On the contrary,
when iron demand is high, the absorbed ferrous iron is transported across the basolateral
membrane into blood. This phase is controlled by ferroportin 1 (FPN1), a ferrous iron
export protein that modulates how much of the enterocyte iron is absorbed into
circulation and made available to the body.FPN1 expression is tightly regulated by the
hepatic hormone hepcidin. Oxidation of absorbed ferrous iron back to ferric iron is
required for transport by plasma protein transferrin (Tf). This is achieved by hephaestin
(HEPH), a multicopper ferroxidase enzyme anchored to basolateral enterocyte membrane
and coupled to FPN1 which catalyses oxidation of Fe 2+ to Fe3+. Fe3+ then bind to Tf which
has high affinity for Fe (III) and enables iron transport around body.

2.3 Role of bone-marrow in iron metabolism-

Erythropoiesis takes place in bone marrow hence this is the prime iron consumer of our
body. Erythropoiesis begins in the bone marrow once pluripotent stem cells differentiate
into erythroid precursor cells in the presence of the hormone erythropoietin. However,
haemoglobin synthesis is only initiated when the erythroid precursor cells start to
differentiate into erythroblasts, expressing transferrin receptors at this stage and taking up

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iron. The first and the final steps of heme synthesis occur in the mitochondrion, while the
intermediate steps take place in the cytoplasm. The first step, which is essentially rate-
limiting, involves the conversion of glycine and succinyl coenzyme A to -
aminolaevulinic acid (ALA) catalysed by the enzyme ALA synthase. Pyridoxine (vitamin
B6) is the coenzyme for this reaction and is stimulated by erythropoietin and inhibited by
haem. The laevulinate product is exported into the cytoplasm, and the condensation of
two ALA molecules forms a monopyrrole pyrrole ring (porphobilinogen) (PBG) by a
condensation reaction. Four molecules of PBG form hydroxymethylbilane (HMB) tetra
pyrrole macrocycle, which cyclizes to form uroporphyrinogen III. The next step is the
modification of the acetate side chains. The decarboxylation reactions produce
coproporphyrinogen, which enters the mitochondrion to be converted into porphyrinogen
IX, i.e., the sequential oxidative decarboxylation of the two propionate groups of pyrrole
rings to two vinyl groups. The final step in the haem biosynthetic pathway is the
oxidation of porphyrinogen IX to protoporphyrin IX. Meanwhile, mitoferrin-1, an iron
transporter, facilitates iron entry into the mitochondrion. By interacting with the ATP-
binding cassette transporter ABCB10, mitoferrin-1 binds to mitochondrial Ferro chelatase
to form an oligomeric complex. Ferro chelatase catalyses the final step of this synthetic
reaction by inserting iron into protoporphyrin IX to produce heme. Subsequently, heme is
exported into the cytosol via mitochondrial heme exporters and combines with globin
chains formed on ribosomes to generate hemoglobin. With repeated cellular divisions, the
nucleus condenses and is eventually lost, thus resulting in a reticulocyte. Ultimately, the
remaining RNA is also lost from the circulating reticulocytes, which mature into
erythrocytes (Troadec et al., 2011) (Figure 3).

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Figure 3. Scheme of heme synthesis

2.4 Recycling of iron

The life span of bone-marrow is 120 days, the bone marrow produces over 200 billion
new erythrocytes daily, involving about 24mg of iron for heme synthesis and
erythropoiesis. Intestinal absorption varies from 1-2mg which is not enough to meet
24mg requirement of iron. Instead, this requirement is mostly met through iron recycling
by RES macrophages phagocytosing senescent erythrocytes. Indeed, approximately 80%
of plasma iron follows the route from the RES to the bone marrow. The spleen is the
major site for iron recycling with some contribution from the liver and the bone marrow.

Senescent erythrocytes exhibit markers of ageing, such as a decrease in membrane

flexibility. These changes are recognised by the splenic macrophages triggering
erythrocyte phagocytosis. Following phagocytosis, the erythrocytes are included in a
phagolysosome and degraded by hydrolytic enzymes and reactive oxygen species to
release their heme. Thereafter, the enzyme HO-1, in the presence of oxygen, catalyses the
breakdown of heme into iron (II), carbon monoxide, and biliverdin. Subsequently, Fe 2+
ions are transported out of the phagolysosome via the natural resistance-associated
macrophage protein-1 (NRAMP1) and enter the cytoplasm to join the LIP (liver
inhibitory protein) (Soe-Lin et al., 2009).

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Normally, there is some erythrocyte destruction occurring within the vasculature. This
releases haemoglobin into the plasma, which immediately binds to the plasma protein
haptoglobin. Upon binding, a haemoglobin–haptoglobin complex is formed, and this
inhibits the oxidative activity of haemoglobin. Monocytes and macrophages, and
particularly splenic and hepatic ones, exclusively express the CD163 protein receptor.
This receptor recognises the haemoglobin–haptoglobin complex and binds to it with high
affinity. Then, CD163-mediated endocytosis of the complex takes place, and its
degradation follows within the macrophage releasing haemoglobin. A wide range of cell
types, including macrophages, possess the low-density lipoprotein receptor-related
protein LRP/CD91 that serves as a receptor for the heme–hemopexin complex and causes
its endocytosis to further contribute to iron recycling.

2.4 Role of liver in iron homeostasis

Liver is the major site of synthesis of hepcidin, a master regulator of iron homeostasis.
Hepcidin is the main negative regulator of iron homeostasis. It acts by binding to FPN1 to
induce its ubiquitination. Subsequently, the internalisation and lysosomal degradation of
FPN1 takes place. This down-regulation of FPN1 occurs at the basolateral membrane of
duodenal enterocytes, as well as from the plasma membrane of RES macrophages,
hepatocytes, and the placenta (Qiao et al., 2012).

Under physiological conditions, the expression of hepcidin is tightly regulated in order to

maintain normal body iron levels. In cases of increased iron levels, hepcidin synthesis is
augmented in an effort to reduce intestinal iron absorption, as well as to reduce the release
of iron from macrophages of the RES into the bloodstream and thus bring iron levels
down to normal. In contrast, when there is iron deficiency, hepcidin production decreases,
and this results in increased intestinal iron absorption, as well as increased iron release
from the RES, which increases the quantity of iron in the circulation. Similarly, in
conditions of anemia and increased erythropoietin activity, hepcidin expression is
suppressed, which increases systemic iron levels (Pantopoulos et al., 2012).

Infection and inflammation trigger an increase in hepcidin synthesis, which serves an

important protective function. The reduction in iron levels reduces the growth of iron-
dependent micro-organisms and helps to eliminate the infection more rapidly. Exposure
of the immune system to pathogens stimulates the release of pro-inflammatory cytokines.

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One of these is interleukin-6 (IL-6). IL-6 is recognised by receptors on the hepatocellular

membrane, and this interaction activates Janus kinases, which phosphorylate signal
transducers and activators of transcription (STAT) proteins, mainly STAT3.
Phosphorylated STAT3 then enters the nucleus, where it binds to HAMP promoter to
stimulate HAMP transcription and subsequent hepcidin production. In conditions such as
chronic infections, inflammatory diseases, and some malignancies, hepcidin levels remain
constantly elevated. This causes a prolonged reduction in intestinal iron absorption, as
well as sequestration of iron in RES macrophages. Moreover, in response to
inflammation, macrophages synthesise small amounts of hepcidin, which targets FPN1 in
an autocrine mode, promoting iron retention within the macrophages. Therefore, less iron
is available for bone marrow utilisation, resulting in anemia of chronic disease.

Cellular iron balance mechanisms also exist for the control of the stability and the
translation of specific iron-related messenger ribonucleic acids (mRNAs) that code for
ferritin, transferrin, TfR1, DMT1, and ALA synthase. Cytoplasmic iron-regulatory
proteins (IRPs) bind to specific iron-responsive elements (IREs) on these mRNAs and
determine whether these undergo translation or not. IRP–IRE interaction promotes the
stability and the translation of the mRNAs for transferrin, TfR1, DMT1, and ALA
synthase. However, in the case of ferritin mRNA, IRP–IRE interaction inhibits its
translation and ferritin synthesis. In iron deficiency, IRPs bind to target mRNA-IREs, and
this interaction increases the synthesis of all the aforementioned proteins, except ferritin,
whose synthesis is decreased. This leads to increased intestinal iron absorption via
DMT1. Moreover, the increase in transferrin and TfR1 promotes cellular uptake of iron
from the circulation, while the decrease in ferritin prevents iron storage. Overall, these
changes enable increased cellular utilisation of iron. In contrast, when there is excess
cellular iron, the binding capacity of IRPs for IREs is lost. This causes mRNA
degradation and diminished synthesis of the above-mentioned proteins, although ferritin-
mRNA stabilisation and enhanced ferritin production occur. Consequently, this reduces
intestinal iron absorption, decreases further TfR1-mediated iron internalisation, and
favours the storage of excess intracellular iron into ferritin (Pantopoulos, K et al., 2012).

Table 2: Hepcidin levels and other iron-related parameters in various types of

anemia (Guido D’Angelo; 2013).

Types of anemia Hepcidin Transferrin Ferritin Soluble

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level saturation transferrin

receptor

Iron deficiency Low Low Low High

anemia (IDA)

Iron-refractory High Low Low Variable

iron deficiency
anemia (IRIDA)

Anemia with iron Low (not High High Variable

overload transfused)

Anemia of chronic High Low Normal or Normal

disease/inflammatio High
n (ACD)

Mixed anemia Normal Low Normal or Normal or

High High

Anemia in chronic High Variable Variable Variable

kidney disease
(ACKD)

Resistance to High Variable Variable Variable

erythropoietin

2.5 Iron refractory anemia-

Iron-refractory iron deficiency anemia (IRIDA) is an autosomal recessive disease

characterized by congenital hypochromic microcytic anemia, low transferrin saturation,
low serum iron, normal–high serum ferritin, and increased hepcidin. This disease is
caused by loss-of-function mutations in TMPRSS6 that lead to high hepcidin and result in
severe anemia.

Role of TMPRSS6/matriptase-2 in iron regulation and anemia

Matriptase-2, encoded by the TMPRSS6 gene, is a member of the type II transmembrane

serine protease family. TMPRSS6 is expressed mainly in the liver and negatively

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regulates the production of hepcidin, the systemic iron regulatory hormone (Chia-Yu
Wang et al., 2014).

Biochemistry of matriptase-2

Type II transmembrane serine protease matriptase-2, encoded by the TMPRSS6 gene,

belongs to the family of type II transmembrane serine proteases (TTSP). Matriptase-2 is
comprised of a transmembrane domain, followed by a sea urchin sperm protein,
enteropeptidase and agrin (SEA) domain, a stem region containing two complement
factor C1r/C1s, urchin embryonic growth factor and bone morphogenetic protein (CUB)
domains and three low-density lipoprotein receptor (LDLR) class A repeats, and a C-
terminal trypsin-like serine protease domain. Matriptase-2 is synthesized as a single chain
inactive proenzyme, which auto-activates itself by a cleavage at an arginine residue at the
RIVGG consensus site between the prodomain and the catalytic domain. After the auto-
activation, it remains membrane-bound through a single disulphide bond linking the pro-
and catalytic domains (Ramsay et al., 2008). Once the catalytic domain is released, it
migrates as a single or dimeric. Matriptase-2 shares high structural and enzymatic
similarities with matriptase-1, which contains four LDLR repeats instead of three, is
expressed in epithelial cells, and has been implicated in the progression of cancers, such
as breast, prostate, and colorectal cancer (Riddick et al., 2005).

The structural features of matriptase-2 are highly conserved across mammalian species
(Figure 4), including human, macaque monkey, dog, cow, mouse and rat, with human
protein sharing >80% identity to matriptase-2 from other species. The expression pattern
of TMPRSS6 determined from mRNA expression studies and analysis of GenBank
Unigene database indicates that matriptase-2 is predominantly expressed in the liver but
also to a lower extent in the kidney, spleen, brain, lung, mammary gland, testis, and
uterus (Ramsay et al., 2008). In addition, aberrant expression of TMPRSS6 is observed in
different human cancers such as breast and prostate cancer (Sanders et al., 2008).

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Figure 4: Schematic representation of matriptase-2, encoded by the TMPRSS6

gene. N: amino-terminus, C: carboxy-terminus, TM: transmembrane domain; SEA: sea
urchin sperm protein, entero peptidase agrin, CUB: complement protein subcomponents
C1r/C1s, urchin embryonic growth factor and bone morphogenic protein 1 domain, L:
low density lipoprotein receptor class A domain (LDLR), Serine Protease: serine protease
domain, red triangle: cleavage activation site (Finberg et al., 2008).

Role of Matriptase-2 in iron metabolism

Matriptase-2 is produced mainly by the liver and negatively regulates the production of
hepcidin, the systemic iron regulatory hormone encoded by the HAMP gene (Finberg et
al., 2008). Hepcidin is a peptide secreted by the liver that plays a central role in adjusting
iron absorption to meet iron needs of the body. Hepcidin negatively regulates cellular iron
export by promoting the degradation of ferroportin (Nemeth et al., 2004), the only known
iron exporter present on the surface of duodenal enterocytes, macrophages, and
hepatocytes and thus limits iron absorption and iron release. It is now well established
that Hamp expression is regulated by the bone morphogenetic protein (BMP)/sons of
mothers against decapentaplegic (SMAD) signalling pathway (Babitt et al., 2007).

At the molecular level, BMP6, the endogenous ligand of BMP/SMAD signalling,

activates BMP-receptor complex by binding to type I and type II BMP receptors that
induces phosphorylation (Andriopoulos et al., 2009). The activated complex, in turn,
phosphorylates Smad1, 5, 8/Smad4 complex, which then translocate to nucleus to
modulate gene transcription. Hemojuvelin (HJV) acts as a coreceptor and is required to
fully activate the BMP signalling ability. The expression of BMP6 is proportional to
hepatic iron concentrations and consistent with Hamp mRNA expression (Kautz et al.,
2009) (Fugure 5).

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Fig. 5: Schematic model of hepcidin regulation by matriptase-2. In conditions of iron

deficiency, matriptase-2 modulates hepcidin signalling by cleaving HJV from the
hepatocyte plasma membrane, resulting in decreased hepcidin production (Silvestri et al.,
2008).

2.6 TMPRSS6 MUTATIONS IN MICE AND HUMAN

Matriptase-2 regulates Hamp expression through the BMP/SMAD pathway in an as yet

unfully characterized manner. Mice without functional matriptase-2 (both mask mice with
truncated Tmprss6 lacking the protease domain and Tmprss6 knockout mice) showed a
hypochromic microcytic anemia and an alopecia. These phenotypes resulted from
inappropriately high levels of Hamp mRNA expression (Finberg et al., 2011).

Mutations in TMPRSS6 in humans led to iron-refractory iron deficiency anemia (IRIDA)

that is unresponsive to oral iron treatment and only partially responsive to parental iron
therapy. IRIDA is also characterized by congenital hypochromic, microcytic anemia, low
mean corpuscular erythrocyte volume, low transferrin saturation, and defects in iron
absorption and utilization (Guillem et al., 2008). Currently, there are 42 different
TMPRSS6 mutations reported in humans, scattered throughout all the different
extracellular domains (Figure 6).

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Fig.6: TMPRSS6 gene structure and schematic representation of corresponding

matriptase-2 mutations reported in IRIDA patients. The genomic organization and
the corresponding structural domains of matriptase-2 with currently identified
mutations are shown. The missense, nonsense, frameshift and splice junction
mutations are shown in green, blue, red and purple arrows, respectively. One in-frame
deletion is boxed in grey. The mutations highlighted in red represent those appear to
have haploinsufficiency (Wang et al., 2014)

Haploinsufficiency is also observed in animal models. It has been reported that Tmprss6
heterozygous knockout mice are more susceptible to iron deficiency compared to their
wild-type littermates (Finberg et al. 2011). It has also demonstrated that, compared to
mice deficient for Hfe alone, heterozygous loss of Tmprss6 in Hfe knockout mice had
higher hepcidin levels at 4 weeks of age, which presumably resulted in decreased hepatic
iron concentrations at 8 weeks of age.

Human genome wide association studies (GWAS) highlighted the significance of

matriptase-2 in control of iron homeostasis by identifying common TMPRSS6 variants
associated with abnormal haematological parameters, including haemoglobin, transferrin
saturation, erythrocyte mean cell volume (MCV) and serum iron concentrations (Tanaka
et al., 2010). Following GWAS, population-based cohort studies were investigated in
China and Italy to study the association between serum iron parameters, iron-related
diseases and specific TMPRSS6 single nucleotide polymorphisms (SNPs): rs855791
(V736A) and rs4820268 (D521D). It was found that TMPRSS6 SNPs was associated
with lowered serum iron, haemoglobin, and plasma ferritin levels, consistent with
lowered risk of iron overload and increased risk of iron deficiency anemia in Chinese
population (An et al., 2012).

2.7 FUNCTION OF MATRIPTASE-2 IN HEPCIDIN REGULATION

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Matriptase-2 inhibition of hepcidin activation by cleaving membrane hemojuvelin has

been established in vitro. When overexpressed in HeLa cells, matriptase-2 interacts and
induces the cleavage of membrane hemojuvelin at the cell surface, resulting in the
generation of soluble hemojuvelin that is released into the cell medium (Silvestri et al.,
2008). However, in both mask and Tmprss6 knockout mice, hepatic hemojuvelin levels at
the membrane were found unexpectedly to be decreased, compared to wild-type animals
(Frydlova et al., 2013). In addition, the levels of serum soluble hemojuvelin, which one
would expect to be decreased in Tmprss6 knockout, did not differ from wild-type mice
(Chen et al., 2013). Although the possibility that soluble hemojuvelin and fragments are
rapidly degraded in vivo cannot be excluded, these data suggested that hemojuvelin may
not be the endogenous substrate of matriptase-2 and that matriptase-2 functions in a more
complicated way in vivo than by merely cleaving hemojuvelin to regulate hepcidin and
iron.

Several studies have been conducted to study the role of matriptase-2, by crossing
Tmprss6 knockout mice with several iron overload mouse models, including the
generations of Hjv/Tmprss6, Bmp6/Tmprss6, Hfe/Tmprss6, and Tfr2/Tmprss6 double
mutant mice. In mice lacking both Hjv and Tmprss6, Id1, a target gene of BMP6
signalling, and Hamp mRNA levels were low, whereas serum iron, transferrin saturation,
and liver iron concentration were high, similar to phenotypes of mice deficient for Hjv
alone (Finberg et al., 2011). These results indicate that if the substrate of matriptase-2 is
downstream of hemojuvelin, it is likely to be along the SMAD signalling pathway. It is
known that inflammatory cytokines, such as LPS and IL6, can induce Hamp expression in
the absence of Hjv, presumably via the Stat3 and Stat5 pathways (Meynard et al., 2013).
However, it was surprising to find that the lack of both Hjv and Tmprss6 in mice did not
impair the responsiveness of hepcidin to BMP2 and IL6, but did fail to respond to iron
challenge (Truksa et al., 2009). In mice deficient for both Bmp6 and Tmprss6, the levels
of Hamp and Id1 mRNAs did not differ from mice deficient for Bmp6 alone; however,
their plasma iron levels and hepatic iron stores were significantly lower, suggesting the
loss of matriptase-2 ameliorates iron overload conditions in Bmp6 knockout mice (Lenoir
et al., 2011). It is unclear why Bmp6/Tmprss6 mice had less iron loading compared to
mice deficient for Bmp6 alone, but Hamp mRNA levels did not differ between
Bmp6/Tmprss6 and Bmp6 knockout mice. Whether matriptase-2 has a significant role
besides effects on BMP/SMAD signalling in iron metabolism, remain to be investigated.

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Mice deficient for Hfe or Tfr2 alone also develop iron overload phenotypes with
inappropriately low Hamp mRNA expression and high serum iron parameters, compared
to wild-type animals (Wallace et al., 2005). It is suggested that Hfe competes with
transferrin for binding to transferrin receptor-1 and thus inhibits Hamp expression
(Schmidt et al., 2008). Others also showed that Hfe knockout mice had high Bmp6
mRNA expression but inappropriately low Smad1/5/8 phosphorylation, suggesting Hfe
facilitates signal transduction initiated by BMP6 (Kautz et al., 2009). However, the
underlying mechanisms of how Hfe and Tfr2 contribute in BMP/SMAD signalling
pathway is unclear. Mice deficient for both Hfe or Tfr2 and Tmprss6, had high Hamp
mRNA expression and exhibited iron deficiency microcytic anemia mimicking the
phenotypes of mice lacking functional matriptase-2 alone (Lee et al., 2012a). This
suggests that Hfe and Tfr2, if involved in BMP/SMAD pathway, are likely to be upstream
of matriptase-2 signalling.

2.8 REGULATION OF MATRIPTASE-2

In rats under acute iron deprivation, hepatic matriptase-2 protein levels are upregulated to
repress hepcidin production (Zhang et al., 2011). Interestingly, matriptase-2 levels are
also increased in response to chronic iron treatment and BMP6 administration in mice,
possibly to prevent excessive hepcidin production, suggesting a dual role of matriptase-2
in the maintenance of tight systemic iron balance in response to iron (Meynard et al.,
2011). In addition, studies also suggest that TMPRSS6 mRNA expression is suppressed
by conditions of inflammation (Meynard et al., 2013) and is upregulated in hypoxia
(Maurer et al., 2012) and by erythropoietin (Peng et al., 2010).

2.9 Mutations in TMPRSS6 cause iron-refractory iron deficiency anemia (IRIDA)

It has been found in one study families with multiple individuals with iron deficiency
anemia unresponsive to oral iron therapy but partially responsive to parenteral iron
administration, suggesting that some cases of iron deficiency may be genetically
determined. The key feature of iron refractory iron deficiency anemia are- a congenital
hypochromic, microcytic anemia, a very low mean corpuscular erythrocyte volume, a low
transferrin saturation, abnormal iron absorption characterized by no haematological
improvement following treatment with oral iron, and abnormal iron utilization
characterized by a sluggish, incomplete response to parenteral iron.

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To determine the genetic basis of IRIDA, they have studied five multiplex kindreds.
Acquired causes of iron deficiency and other inherited causes of microcytosis were
rigorously excluded. It has been analysed all TMPRSS6 coding regions and intron–exon
boundaries and identified sequence variants in each of the five multiplex IRIDA kindreds.
Affected individuals harboured frame-shift mutations, splice junction mutations or
missense mutations altering residues conserved in TMPRSS6 homologs in humans. In
three of the four kindreds in which the phase of chromosomal segregation was known,
they identified bialleic mutations. In the fourth family, they found a mutation only on the
paternal allele; however, they did not exclude the presence of other types of mutations,
such as large deletions, that would not be detectable by sequencing. Additionally, in the
fifth kindred, for which DNA was available from only the affected individuals, they
found a non-conservative missense mutation in both siblings. They also examined two
individuals with sporadic IRIDA and found nonsense, frameshift or splice junction
mutations in both. These findings conclusively show that mutations in TMPRSS6 cause
IRIDA (Figure 7).

Figure 7: Schematic representation of TMPRSS6 mutations and corresponding

TMPRSS6 domains. The missense, nonsense, frameshift and splice junction mutations
present in five familial and two sporadic cases of IRIDA are diagrammed adjacent to the
affected TMPRSS6 domain. The transmembrane (TM), complement factor C1r/C1s,
urchin embryonic growth factor and bone morphogenetic protein (CUB), LDL-receptor
class A (L) and trypsin-like serine protease domains are shown in green, blue, yellow and
red, respectively (Finberg KE et al., 2008).

3. Endoplasmic reticulum stress

The endoplasmic reticulum (ER) is a vital organelle present in all eukaryotic cells. It
consists of interconnected, branching membranous tubules, vesicles, and cisternae that
provide a distinct subcellular compartment with a number of functions. The rough ER is

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studded with ribosomes on its outer surface and plays a key role in protein synthesis and
secretion. The smooth ER lacks associated ribosomes and therefore is not primarily
involved in protein synthesis, but is central to the synthesis of fatty acids and
phospholipids, assembly of lipid bilayers, metabolism of carbohydrates, and regulation of
calcium homeostasis.

Due to the cytotoxic risk that the accumulation of misfolded/unfolded proteins poses to
the cell, it is not surprising that cellular sensors and pathways have evolved to respond to
this threat. This stress response system, called the ER stress response or the UPR,
displays a dichotomic yin-yang characteristic, where mild or short term stress triggers
activation of a response module that either leads to the neutralization of the initial stress
or adaption to it, but where severe or long-lasting stress favours activation of a
proapoptotic module that will lead to cell death. In both cases, the initial signal (e.g.,
accumulation of misfolded proteins inside the ER) is transmitted across the ER
membrane, through the cytoplasm, and into the nucleus, where alterations in gene
expression patterns effect the respective resulting phenotypic outcome, that is, adaptation
and survival or apoptosis (Figure 8).

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Figure 8: Triggers of ER stress and the yin-yang balance of cell survival versus cell
death. A great variety of conditions and pharmacological compounds can disturb ER
homeostasis, leading to ER stress and the accumulation of unfolded and misfolded
proteins. In response, ER stress signalling pathways stimulate pro-survival efforts to
either neutralize the stressful insult or adapt to it. GRP78 plays a key role in the cell’s
attempt to adapt and survive. In contrast, if ER stress is too severe, the pro-apoptotic
module of this cellular system gains dominance and shifts the balance towards cell death.
CHOP represents a central executor of this latter process. In essence, these opposing
processes of cell death versus survival are reflective of the yin-yang (shadow and light)
concept of Chinese philosophy, where seemingly contrary forces are interconnected and
interdependent as part of a greater whole. Although many other components participate in
balancing the cell’s yin-yang response to ER stress, the opposing efforts of prosurvival
(yang) GRP78 and proapoptotic (yin) CHOP represent important tenets of this struggle;
as well, their expression levels are being used as convenient markers and readouts as to
the ER stress status of cells.

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3.1 Key Players in the ER Stress Response

The diversity of functions of the ER, which include quality control and secretion of
proteins, lipid and membrane biosynthesis, and control of intracellular calcium
homeostasis, it is not unexpected that a large number of regulatory components
participate in these processes and, in one way or another, become involved in the ER
stress response/UPR. For example, the ER lumen is rich in calcium dependent molecular
chaperones, such as glucose-regulated protein 78 (GRP78, also called BiP:
immunoglobulin heavy chain-binding protein, GRP94, calnexin and calreticulin, enzymes
involved in posttranslational protein modifications, such as protein disulphide isomerase
(PDI), oxidoreductases, and those performing protein glycosylation and lipidation, as well
as numerous others involved in lipid and membrane biosynthesis.

Master Regulator GRP78- Among the many ER-resident proteins, the chaperone
GRP78 stands out because in addition to its calcium binding and protein processing
function, it exerts a key role as a master initiator of early ER stress/UPR signalling. As
implied by its name, GRP78 initially has been characterized as a glucose-regulated
protein, where restricting the availability of glucose in cell culture medium resulted in
pronounced stimulation of GRP78 transcription and translation, and thus provided initial
clues as to its activation during cellular stress conditions. A large number of subsequent
studies established that a great variety of cellular and micro environmental disturbances,
as well as many pharmacological interventions, can lead to increased GRP78 expression,
along with aggravated ER stress. Indeed, the significantly increased amount of GRP78
protein over baseline expression has become an established indicator and marker for the
presence of cellular ER stress. GRP78 belongs to the heat shock protein 70 (HSP70)
family of proteins, where many of its members have been characterized as chaperones
within the ER. In recent years, however, it was discovered that GRP78 can also be present
outside the ER; for example, the protein was found in the cytosol, in mitochondria, in the
nucleus and at the cell surface of tumour cells.

In unstressed cells, a fraction of ER-luminal GRP78 is bound to three different ER

transmembrane proteins:

(i) inositol-requiring kinase/endoribonuclease 1 (IRE1)

(ii) protein kinase activated by double-stranded RNA (PKR)- like ER kinase
(PERK) and

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(iii) Activating transcription factor 6 (ATF6).

Binding of GRP78 to the ER-luminal domains of these proteins keeps their activity
suppressed and maintains them in an inactive state. Upon ER stress and concomitant
accumulation of misfolded and unprocessed proteins, GRP78 is sequestered away from
PERK, IRE1, and ATF6 in order to attend to the increased need for protein folding. As a
result, dissociation from GRP78 leads to the activation of all three of these
transmembrane proteins, thereby unfolding three distinct branches of the ER stress
response/UPR. Among the consequences of these signalling events is increased
expression of GRP78, which not only serves to provide the needed additional chaperone
capacity, but also eventually will reassociate with PERK, IRE1, and ATF6 in order to
return these signalling modules to their inactive modes when homeostasis has been re-
established (Figure 9).

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Figure 9: Overview of the three signalling branches of the ER stress response/UPR.

In the absence of ER stress, ER luminal GRP78 associates with ER transmembrane
proteins PERK, IRE1, and ATF6 to block their activation (shown as inactive UPR in the
top right square). Upon ER stress, accumulating unfolded and misfolded proteins inside
the ER sequester GRP78, thus dissociating this master regulator from all three
transmembrane sensors and relieving their blockage. Activation of PERK entails
homodimerization and auto phosphorylation, leading to phosphorylation of eIF2𝛼, which
terminates global protein translation. , but exempts selected ER stress-associated proteins,
such as ATF4. Activation of IRE1 also entails homodimerization and auto
phosphorylation. Endonuclease activity of activated IRE1 removes an intron from Xbp1
mRNA to generate a shorter splice variant that encodes transcription factor XBP1. ATF6
translocates to the Golgi, where it is proteolytically cleaved by S1 and S2 proteases to
generate the transcriptionally active p50 fragment. All three transcription factors, ATF4,
XBP1, and ATF6-p50 translocate into the nucleus where they regulate the expression of a
variety of gene products collectively involved in managing ER stress

3.2 The IRE1 Signalling Branch-. Activation of IRE1 represents the most conserved
signalling branch of the ER stress response/UPR. It is a bifunctional enzyme with serine/
threonine protein kinase and endoribonuclease (RNase) activity in its cytosolic domain.
Release from suppression by GRP78 triggers its homodimerization and auto
phosphorylation as part of the activation process (Parmar and Schroder 2012). Activated
IRE1 cleaves a 26-base fragment from the mRNA encoding X box binding protein 1

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(XBP1), resulting in spliced XBP1s and translation of a potent transcription factor

controlling the expression of genes involved in ERAD and protein folding, as well as
others directing the synthesis of phospholipids that are required for the expansion of ER
membranes during ER stress. IRE1 signalling and XBP1 splicing are particularly
important in highly secretory cells where the protein folding machinery is continuously
engaged with a high amount of nascent proteins. Therefore, this branch of control serves
as a key adaptive mechanism to match ER folding capacity with the demands of protein
folding. In addition to splicing a number of mRNAs, a second function of IRE1 is to
activate a signalling cascade involved in controlling cell fate with regard to cell death.
Here, activated IRE1 recruits tumour-necrosis- factor-receptor-(TNFR-) associated factor
2 (TRAF2), which results in the downstream activation of apoptosis signal-regulated
kinase1 (ASK1) and c-Jun N-terminal kinase (JNK) (Nishitoh et al., 2002). On one hand,
sustained JNK activity during prolonged ER stress inhibits antiapoptotic members of the
Bcl-2 (B cell lymphoma 2) family of proteins. On the other hand, JNK phosphorylates
and activates proapoptotic BH3-only proteins, such as Bid (BH3 interacting domain death
agonist) and Bim (Bcl-2-interacting mediator of cell death). Combined, these events lead
to oligomerization of Bax and Bak, resulting in permeabilization of the outer
mitochondrial membrane and execution of the intrinsic apoptotic process.

3.3 The ATF6 Signalling Branch- ER transmembrane-localized ATF-6 harbours a basic

leucine zipper (bZIP) motif and transcription factor properties. Upon its release from ER
luminal GRP78, Golgi localization sequences are unmasked, where upon ATF6
translocate to the Golgi apparatus. Here, it is proteolytically cleaved by Golgi-resident
site-1 protease (S1P, a serine-protease) in its ER luminal domain and by site-2 protease
(S2P, a metalloprotease) within its region that spans the Golgi phospholipid bilayer,
resulting in the release of the cytosolic bZIP transcription factor domain from the Golgi
membrane (Haze et al., 1999). Upon translocation to the nucleus, ATF6 stimulates
expression of a number of genes whose protein products contribute to protein folding,
protein secretion, and ERAD, thereby supporting the cell’s effort to cope with ER stress
and accumulated misfolded/unfolded proteins. Examples of ATF6-regulated genes
include GRP78 and GRP94, protein disulphide isomerase (PDI), XBP1, and CHOP.

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3.4 The PERK Signalling Branch- Activation of PERK involves its homodimerization
and auto phosphorylation, which is followed by phosphorylation of its main substrate,
eukaryotic initiation factor 2 alpha (eIF2𝛼). Phosphorylation of eIF2𝛼 attenuates global
protein synthesis, thereby decreasing protein influx to the ER in support of resolving the
cytotoxic threat from accumulated misfolded proteins (Harding et al., 2000). At the same
time, phosphorylation of eIF2𝛼 changes the efficiency of AUG initiation codon usage and
leads to the preferential translation of a small number of mRNAs, including activating
transcription factor 4 (ATF4), a transcription factor that stimulates a set of genes involved
in supporting recovery and adaptation. Among ATF4-regulated genes is the one encoding
CHOP, a key transcription factor that is important to initiate the apoptotic program in
case of excessive ER stress. Besides, eIF2𝛼, nuclear factor-erythroid related factor 2
(Nrf2) represents a second immediate substrate for phosphorylation by PERK. Upon
activation, this basic leucine zipper transcription factor migrates to the nucleus where it
activates genes encoding antioxidant proteins and detoxifying enzymes (Cullinan and
Diehl, 2004). Because ER stress may involve the accumulation of reactive oxygen species
(ROS), thereby promoting a state of oxidative stress, Nrf2 plays a critical role in fighting
such perturbations in redox homeostasis (Cullinan and Diehl, 2006).

3.5 Pro-apoptotic CHOP- The expression levels of CHOP (C/EBP homologous protein,
also called GADD153: growth arrest and DNA damage inducible gene 153) are kept very
low in non-stressed cells. Upon acute ER stress, however, CHOP expression is strongly
stimulated through IRE1- and PERK-mediated signalling and the activities of ATF4 and
ATF6 transcription factors. The full pro-apoptotic effect of CHOP only emerges when ER
stress cannot be subdued by the efforts of the pro-survival module of the response system,
and the levels of misfolded proteins remain high. In this case, CHOP stimulates a
transcriptional profile that facilitates a pro-apoptotic program. It includes expression of
pro-apoptotic Bim and repression of anti-apoptotic Bcl-2, which represents a mechanism
that is aligned with similar pro-apoptotic efforts of JNK mentioned previously (H.
Nishitoh et al., 2012). As well, CHOP induces death receptor 5 (DR5), which further
sensitizes cells to apoptotic stimulation by a variety of conditions that cause ER stress (H.
Yamaguchi et al., 2004). Another target gene of CHOP is growth arrest and DNA damage
inducible protein 34 (GADD34), a regulatory subunit of protein phosphatase type 1
(PP1); CHOP induced stimulation of GADD34 expression leads to PP1 activation and
dephosphorylation of eIF2𝛼, resulting in the resumption of general translation and normal

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functioning. Thus, while sustained elevation of CHOP expression triggers strong pro-
apoptotic signalling, its initial effect on GADD34 may contribute to the restoration of
homeostasis—with the caveat that the renewed supply of client proteins to the ER, if
taking place too early, that is, under conditions where ER stress is not yet completely
resolved, can trigger the generation of reactive oxygen species (ROS) with deleterious
consequences for cell survival (Malhi and Kaufman, 2011) (Figure 10).

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Fig. 10: Cell death signalling by the ER stress response/UPR. In case of severe and
sustained ER stress, a number of pro-apoptotic events begin to dominate and lead to
apoptosis. Transcription factors ATF4 and ATF6-p50 stimulate CHOP expression. On
one hand, CHOP stimulates expression of GADD34, which associates with PP1, resulting
in dephosphorylation of eIF2𝛼𝛼, thus reactivating global cellular protein synthesis. On
the other hand, CHOP inhibits anti-apoptotic proteins of the Bcl-2 family and stimulates
pro-apoptotic Bim, altogether leading to heterodimerization and activation of pro-
apoptotic Bax and Bak. CHOP also stimulates expression of cell surface death receptor
DR5, which sensitizes cells to pro-apoptotic stimuli, presumably via calibrating the
extrinsic apoptotic pathway involving caspase 12. Similarly, activated JNK complements
the pro-apoptotic efforts of CHOP. JNK becomes phosphorylated and activated by protein
kinase ASK1 upon association of TRAF2 with activated IRE1. Association of TRAF2
with activated IRE1 also leads to activation of caspase 12. Calcium release from the ER
via IP3 receptors can activate calpains, which further stimulate caspase 12 activation via
proteolytic cleavage of its inactive procaspase precursor.

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