disorders of calcium

8/8/2019 Disorders of Calcium

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In the name of ALLAH, the Beneficent the Merciful


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DISORDERS OF CALCIUM

MUHAMMAD KHAWAR NAZIR


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INRODUCTION

Keratinocytes and CaIntercellular communication

Intercellular adhesion

Intracellular signaling pathways

Differentiation

Ca pumps

MitochondrialEndoplasmic reticulum (ER) and Golgi aparatus

Cell membrane


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Calcium homeostasis

Genes that encode Ca-ATPases:

ATP2A1 SERCA1

ATP2A2 SERCA2

ATP2A3 SERCA3

Sarco (endo) plasmic-reticulum

Ca2-ATPase

Transport Ca from cytosol into ER

lumen

SERCA2a Slow cardiac muscle, skeletal and neonatal muscle.

Much lower levels in non-muscle cell like keratinocytes.

SERCA2b Principal Ca ATPase in smooth muscle and non muscle tissuelike keratinocytes.

Higher expression in basal keratinocytes.

SERCA3 Intestine, lung and spleen but not in keratinocytes or hair follicles.


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Calcium homeostasis

Genes that encode Ca-ATPases:

ATP2C1 SPCA1

ATP2C2 SPCA2

Secretory pathway Ca/Mn- ATP

ase protein 1 and 2

Transport Ca from cytosol into Golgi

aparatus (trans face)

ATP2C1 mRNA Present in all tissues

Vital housekeeping function

ATP2C2 mRNA Along gastrointestinal tract


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Calcium homeostasis

Why mutations on essential enzymes for many cell

types causes predominantly skin-disease?

Compensatory capability

SERCA2+/- mice showed high

adaptability of Ca signaling and

dependant cellular functions in vivo

Human keratinocytes cant compensate

lack of SERCA2 .Lack of SERCA3 in

them?

SERCA and SPCA involved in

Golgi Ca uptake

Human keratinocytes depend

mostly on SPCA1.

Hailey-hailey only has

cutaneous involvement.


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Calcium homeostasisStructure and function

3 major parts:

Cytoplasmic headpiece More than half the structure

Phosphorylation and nucleotide binding domain

Active site for ATP hydrolysis

Stalk domain

Transmembrane domain Anchor to the lipid bilayer

Ca binding sites (2 domains in SERCA and 1 Ca/Mn SPCA)

Transmembrane channel for Ca passage.

Ca binds to transmb domain accessible only from cytoplasm

Phosphorylation Ultra structural changes

Access from cytoplasm is no longer available

Access to the lumen is gained and Ca affinity lost, releasing in to the

lumen


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Calcium pumps and desmosome adhesion

Desmosome integrity Transmembrane cadherins prot (Deg, Dsc)Submembranous plaque prot (PG, PF, DP)

Keratin filaments

Adhesion Ca dependant interactions between extracell Cadherins

Assembly Low Ca conditions, desmosomes are not assembled but constituentproteins are synthesized


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Calcium pumps and desmosome adhesion

Protein synthesis Synthesis, processing, and maturation of constituentproteins is Ca and Mn - level dependant in the lumen ofER

and Golgi (SERCA2b, SPCA1 respectably)

Mutations in ATP2A2 Expression, degradation and activity of SERCA

Inactive or reduced by 50% in Dariers disease.

Most mutations markedly reduce expression by enhancing

proteasome mediated degradation.

Apoptosis Loss of desmosomal adhesion triggers ANOIKIS, a type ofapoptosis characterized by cell detachment.

Changes in Ca transport activity

RE overload response


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Calcium

Major signal for keratinocyte differentiation

Required for desmosome and adherens junction formation

Fourfold increase from basal to cornified layers

In vitro keratinocytes + Ca:

Cell cycle arrest in G1

Expression of K1, 10,

Involucrin, loricrin and filaggrin


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Voltage-dependent calcium

channels Voltage-dependent calcium channels (VDCC) are a group of

voltage-gated ion channels found in excitable cells (e.g., muscle,glial cells, neurons, etc.) with a permeability to the ion Ca2+

At physiologic or resting membrane potential, VDCCs are normally

closed

They are activated (i.e., opened) at depolarized membranepotentials and this is the source of the "voltage-dependent" epithet

Activation of particular VDCCs allows Ca2+ entry into the cell, which

depending on the cell type, results in muscular contraction,excitation of neurons, up-regulation ofgene expression, or releaseofhormones orneurotransmitters


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Structure of VD Calcium Channels

Voltage-dependent calcium channels are

formed as a complex of several different

subunits: 1, 2, 1-4, and

The 1 subunit forms the ion conducting

pore while the associated subunits have

several functions including modulation of

gating


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Calcium Channel Structure

E1 subunit is comprised of four homologousdomains, each of which contains six transmembranehelices

F subunit is intracellular and associates with the E1subunit

K subunit is a glycoprotein that possesses fourtransmembrane segments

E2 subunit is extracellular, highly glycosylated andassociates with the membrane spanning H subunitvia disulfide bonds.


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Calcium Channel Structure

Calcium Channel StructureCalcium Channel StructureCalcium Channel Structure


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Channel Subunits

Several different kinds of high-voltage-gated calcium channels (HVGCCs)

Structurally homologous among varying types; All similar, but notstructurally identical

In the laboratory, it is possible to tell them apart by studying theirphysiological roles and/or inhibition by specific toxins

High-voltage-gated calcium channels include the neural N-type channelblocked by -conotoxinGVIA, the R-type channel (R stands forResistant tothe other blockers and toxins) involved in poorly defined processes in thebrain, the closely related P/Q-type channel blocked by -agatoxins, and thedihydropyridine-sensitive L-type channels responsible for excitation-contraction coupling ofskeletal, smooth, and cardiac muscle and forhormone secretion in endocrine cells.


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1 Subunit

The 1 subunit pore (~190 kDa in molecular mass) is theprimary subunit necessary for channel functioning in theHVGCC, and consists of the characteristic four

homologous I-IV domains containing six transmembrane-helices each

The 1 subunit forms the Ca2+ selective pore, whichcontains voltage-sensing machinery and the drug/toxin-

binding sites

A total of ten 1 subunits that have been identified inhumans


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2 Subunit

The 2 gene forms two subunits 2 and . They are linked to each

other via a disulfide bond and have a combined molecular weight of 170kDa. The 2 is the extracellular glycosylated subunit that interacts themost with the 1 subunit. The subunit has a single transmembraneregion with a short intracellular portion, which serves to anchor the proteinin the plasma membrane. There are 4 2 genes:

(CACNA2D1),

(CACNA2D2),

(CACNA2D3),

(CACNA2D4).

Co-expression of the 2 enhances the level of expression of the 1subunit and causes an increase in current amplitude, faster activation andinactivation kinetics and a hyperpolarizing shift in the voltage dependenceof inactivation. Some of these effects are observed in the absence of thebeta subunit, whereas, in other cases, the co-expression of beta isrequired.

The 2-1 and 2-2 subunits are the binding site for at least twoanticonvulsant drugs, gabapentin (Neurontin) and pregabalin (Lyrica), thatalso find use in treating chronic neuropathic pain.


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Subunit

The intracellular subunit (55 kDa) is an intracellularMAGUK-like protein (Membrane-Associated GuanylateKinase) containing a guanylate kinase (GK) domain andan SH3 (src homology 3) domain

The guanylate kinase domain of the subunit binds tothe 1 subunit I-II cytoplasmic loop and regulatesHVGCC activity. There are four known isoforms of the subunit:

CACNB1

CACNB2

CACNB3

CACNB4


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Subunit

The 1 subunit is known to be associated with skeletal muscle VGCCcomplexes, but the evidence is inconclusive regarding other subtypes ofcalcium channel.

The 1 subunit glycoprotein (33 kDa) is composed of four transmembranespanning helices. The 1 subunit does not affect trafficking, and, for themost part, is not required to regulate the channel complex. However, 2, 3,

4 and 8 are also associated with AMPA glutamate receptors. There are 8 genes for gamma subunits:

1 (CACNG1),

2 (CACNG2),

3 (CACNG3),

4 (CACNG4),

(CACNG5),

(CACNG6),

(CACNG7), and

(CACNG8).


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Acantholytic Disorders of Skin

These advances have also allowed a betterclassification of related localized acantholyticconditions, namely, epidermal dyskeratotic

acantholytic nevi, which are a localized form ofDD caused by somatic mosaicism, andacrokeratosis verruciformis (AKV) of Hopf, whichwas found to be allelic to DD

Thus, defects in the same gene encoding thiscalcium pump of the endoplasmic reticulum (ER)underlie three related conditions.


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A Simplified Representation of

Ca2+ signaling

in keratinocytes


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1. Ca2+ binding to its plasma membrane receptor (CaR) activates phospholipase C (PLC )


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3. IP3 binds to its receptor (IP3R) at the surface of the endoplasmic reticulum (ER) and Golgi apparatus, which

causes the depletion of intracellular stores and induces an increase in intracellular Ca2+ levels


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4. This increase triggers the opening of Ca2+ releaseactivated channels in the plasma

membrane, which leads to a sustained increase in Ca2+ intracellular levels


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5. Ca2+ binds to calmodulin; this activates calcineurin and calmodulin-dependent protein kinases, which

regulate gene transcription through phosphorylation/dephosphorylation of transcription factors


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7. Ca2+ efflux to the extracellular space involves plasma membrane Ca2+ATPases (PMCA)

and Na+/Ca2+ exchangers (NCX)


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8. Mitochondria take up Ca2+ released from the internal stores during Ca2+ signaling via the Ca2+ uniporter and returnit to the cytosol through an NCX.


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Thus, Ca2+ homeostasis requires differential Ca2+ concentrations in the

cytosol, the sarco/endoplasmic reticulum, the Golgi apparatus, the

mitochondria, and the nucleus of the cell. The largest store of cellular Ca2+ is

located in the ER lumen and in Ca2+-binding proteins. Ca2+ signaling is highly

regulated and generated by influx through Ca2+ receptors, release from

internal stores (ER, Golgi apparatus, mitochondrion), and sequestration by

Ca2+ pumps (SERCAs, hSPCA1) and Ca2+ exchangers. mNCX =

mitochondria Na2+/Ca2+ exchanger.


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Darier-White Disease


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Endoplasmic Reticulum

Mutations in ATP2A2gene,

Ca2+ ATPase (SERCA2),


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Darier disease.

Truncal

involvement in

seborrheicareas.

Keratotic

papules can

vary fromred (A)

to

brown (B)

in color and

may becomeconfluent.

Courtesy:

Bolognia:


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Darierdisease.

Severe

involvement inintertriginous

zones,

including the

groin,

submammary

area and

abdominal fold.


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Autosomal dominant disease

Prevalence 1 in 26,300 in Slovenia, 1 in

30,000 in Scotland, 1 in 36,000 innortheast England, and 1 in 100,000 in

Denmark

Penetrance of the disease is complete,

and expression is highly variable


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The gene forDD was mapped by linkage analysis to chromosomeband 12q23-24 in 1993, with no evidence for genetic heterogeneityin affected families

ATP2A2was identified as the defective gene in 1999 using a

candidate positional cloning approach

ATP2A2encodes sarco/endoplasmic reticulum Ca2+ adenosinetriphosphatase (ATPase) isoform 2 (SERCA2), a calcium pumptransporting Ca2+ from the cytosol to the lumen of the ER

The identification of a Ca2+ pump as the defective protein in

DDcame as a surprise and shed light on the key role of Ca2+ signaling

in the homeostasis of the epidermis.


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ATP2A2spans 76 kilobases (kb), is organized in

21 exons, and encodes a 4.4-kb transcript,

which is alternatively spliced into 3 isoforms:

1) SERCA2a,

2) SERCA2b,

3) SERCA2c.


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SERCA2a is expressed in the heart and

slow-twitch skeletal muscles

SERCA2b and SERCA2c are ubiquitously

expressed

SERCA2b is the major isoform detected

in the human epidermis


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SERCA2b is the major isoform

detected in the human

epidermis


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SERCA2 pumps belong to the P-type Ca2+

ATPase family, defined by the highly conserved

phosphorylation sequence DKTGT

They catalyze the hydrolysis of ATP coupled

with the translocation of 2 Ca2+ ions from the

cytosol (100 nM) to the ER lumen, where Ca2+

is stored at high concentrations (500 M)


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SERCA pumps comprise 3 cytoplasmic domains (theactuator, the phosphorylation, and the ATP-bindingdomains) linked by five stalk domains to 10 or 11transmembrane domains anchored in the ER membrane.

After binding of two Ca2+ ions, SERCA pumps undergotrans-phosphorylation from ATP, which leads toconformational changes and the release of Ca2+ ions

into theE

R lumen.

This complex cycle involves critical interactions betweencytoplasmic and transmembrane domains, four of whichform the two Ca2+ ions binding pockets of the molecule.


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The majority of the mutations are missense mutations(50 percent) or in-frame deletions or insertions (8percent), which predict the synthesis of a structurallyabnormal protein.

Other mutations include nonsense mutations (12percent) and frameshift mutations (23 percent), whichlead to a premature termination codon (PTC) and predictloss of protein expression through nonsense messengerRNA (mRNA) decay.

The remaining mutations are splice site mutations (7percent), the effects of which on the mutated proteinremain to be documented.


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Each of these mutations leads to a PTC, which

indicates that loss ofSERCA2b expression is

sufficient to cause DD and that SERCA2a

isoform cannot compensate for it.

This finding is consistent with the fact that

the most prominent expression of SERCA2b

is in the epidermis, although both isoformsare expressed in cultured keratinocytes.


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To provide a better understanding of the physiologicfunctions of SERCA2 in vivo, knockout mice weredeveloped by targeting the promoter and first two codingexons of SERCA2

Homozygous mutants were not observed, consistentwith the idea that SERCA2b serves an essentialhousekeeping function. SERCA2 heterozygous mutantsappeared healthy but showed reduced cardiac musclecontractility and relaxation, and displayed a very high

frequency of squamous cell carcinomas with age

Heterozygous mutant mice did not develop DD-likelesions, however, which reveals significant speciesdifferences in susceptibility to the disease.


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Ca2+ triggers the switch between

keratinocyte proliferation and

differentiation

The increase of intracellular Ca2+ levels

activates calmodulin, a major Ca2+-

binding protein that plays a key role in the

control of gene transcription by Ca2+


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This results in the activation ofthe cytosoliccalmodulin-dependent phosphatase calcineurinand the family of Ca2+/calmodulin-dependentprotein kinases

Both types of enzymes contribute to theregulation of cell division and differentiation.Calcineurin dephosphorylates the cytoplasmic

NFAT (nuclear factor of activated T cells)proteins, which allows their translocation into thenucleus and the induction of their downstreamtarget genes


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It is of interest to note that

cyclosporin A, tacrolimus, and

pimecrolimus are

Calcineurin Inhibitors


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Activation of the Ca2+/calmodulin-dependent proteinkinase cascade involves 3 distinct kinases that regulatetranscription through phosphorylation of nuclear factorsand histone acetylation, which leads to transcriptional

activation or inhibition.

By keeping appropriate Ca2+ concentrations in the ERlumen, SERCA2 also plays an essential role in proteinsynthesis, chaperone-dependent processing, and post-

translational modification of membrane and secretedproteins, which require a unique calcium-richenvironment.


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Ca2+ is required for

1)Assembly of desmosomes

2)Adherens junctions3)Actin polymerization


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A total of 68 ATP2A2 mutations identified in DD

patients have been studied by site-directed

mutagenesis in three separate studies

DD mutations cause markedly reduced protein

expression and/or loss of Ca2+ transport

through defective ATPase activity


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Rare mutant was found to have increased transportactivity but higher affinity. Together with ATP2A2mutations leading to PTCs and loss of expression of themutant allele, these results support the proposition that

haploinsufficiency is a common mechanism for thedominant inheritance ofDD

However, some missense mutations partially inhibit theco-expressed wild-type protein and are associated with a

more severe phenotype,which suggests that somemissense mutations may act at least in part through adominant negative mechanism


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In the same study, inhibition of ATPC1 by smallinterfering RNA diminished DD keratinocyteviability, which indicates that upregulation of

ATP2C1 was a compensatory mechanism

against apoptosis

Pani et al. have shown upregulation of one ofthe plasma membrane capacitive entrychannels, transient receptor potential cationicchannel 1 (TRPC1) in DD and SERCA2+/mouse keratinocytes


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These authors also demonstrated that upregulation ofTRPC1 ( Transient Receptor Potential Channel 1)enhances cell proliferation and restricts apoptosis, whichillustrates anothercompensatory mechanism inresponse to ER Ca2+ store depletion. These results

emphasize the interplay between the ER, Golgiapparatus, and plasma membrane Ca2+ channels suchas TRPC1 in Ca2+ signaling

They indicate that upregulation of other Ca2+ transport

ATPase isoforms (ATP2C1) and channels could at leastin part compensate for ATP2A2 dysfunction in the steadystate and that blockade of these compensatoryresponses compromises cell viability.


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A change in the Ca2+ ER luminal content is likely to have a majoreffect on Ca2+ signaling, protein post-translational modifications,and trafficking

Although no glycosylation defect of the desmosomal proteins wasdetected in DD keratinocytes, in culture trafficking of desmoplakin to

the membrane is significantly hindered

Defective expression of this key molecule that links the cytoskeletonto the desmosomal complexes could contribute to impaired cell-to-cell adhesion in the epidermis in DD.

Finally, Ca2+ plays a key role in the transcriptional regulation of awide range of genes that are essential for epidermal cell-to-celladhesion and terminal differentiation, which suggests that abnormalCa2+ signaling could have a profound impact on the transcription ofthese genes.


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The observation that exposure to ultraviolet B (UVB)irradiation, heat, and infection triggers diseasemanifestations indicates the role of external factors inunmasking the basic defect in DD

In the absence of stress, SERCA2 deficiency can becompensated by increased expression of the normalallele and/or other regulatory systems

Triggering factors would disrupt this subtle balance by

downregulating ATP2A2 or increasing the requirementfor SERCA2 to maintain a unique Ca2+ content in thelumen


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Because only one copy of ATP2A2 is functional, reduction ofSERCA2 levels would be excessive, or compensation would not

reach adequate levels. Consistent with this possibility, UVBirradiation and pro-inflammatory cytokines have been shown todownregulate ATP2A2 as well as ATP2C1 mRNA expression

Reduced levels of SERCA2 would result in inappropriate Ca2+concentrations in the lumen, causing dysfunction of calcium-dependent chaperone molecules that leads to impair folding,assembly, and/or trafficking of proteins. Whether this preferentiallyaffects proteins playing a key role in cell-to-cell adhesion and/ordifferentiation or affects protein processing in general remains to bedetermined.


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Dariers diseaseAutosomal dominant

Malodorus warty papules and plaques

Palmoplantar pits

Nail abnormalities

Hx:

suprabasal acantholysis with clefting

hyperproliferative budding

Dyskeratosis (corp ronds and grains)


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Pathogenesis:..Autosomal dominant ATP2A2 Endoplasmic reticulum Ca ATP ase (SERCA2)

Abnormal intracellular Ca signaling of endoplasmic reticulum andmitochondria

Inadequate filling of the endoplasmic reticulum with Ca

Impaired processing of

desmosomal proteins

Apoptosis

Dyskeratotic keratinocytes

Acantholisis


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Mechanisms leading to apoptosis

Decreased ER Ca levels accumulation of unfoldedproteins

ER overload

response (EOR)

Unfolded protein

response (UPR).

factor NF - B

Transcription of pro-

inflammatory proteins and

cell-adhesion molecules

ER resident stress

proteinsSuppression of

protein synthesis



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Acrokeratosis Verruciformis

Grover Disease

Hailey-Hailey Disease


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Rare, heritable disorder of keratinization,originally described by Hopfin 1931

Usually presents with multiple small flat-toppedpapules predominantly on the dorsum of thehands and feet

Histopathologic analysis shows hyperkeratosis,hypergranulosis, and acanthosis with "churchspikes," circumscribed elevations of theepidermis, without acantholysis or dyskeratosis


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Acrokeratosis

verruciformis

of Hopf:

Involvement

of the dorsal

aspects of the

hands with

flat-topped

papules.


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Allelic with Darier-White disease:Both are due to defects in ATP2A2.


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Autosomal dominant

This mutation is located in the ATP-binding domain ofSERCA2 and has not been reported in patients withclassic DD. Functional analysis showed that it abolishesCa2+ transport

These results established thatAKV and DD can be dueto mutations in the same gene (i.e., are allelic disorders)

However, one study in a large Chinese family did not findevidence for the involvement of ATP2A2, which raisesthe possibility of genetic heterogeneity in AKV



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Although initially viewed as distinct entities,AKV andDD, especially the acral form of the latter, have beenconsidered as related disorders by several authors, evenby Hopf and Darier themselves

In support of AK's being part of the DD spectrum is thedescription of the co-occurrence of AKV and DD in thesame patient or in affected members of the same family

In addition, it is notable that 50 percent of

DDpatientshave acral warty papules and that patients whose

lesions show the histologic changes of AKV have beenreported to develop lesions with typical histologicfeatures ofDD



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Grover Disease



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Grover Disease

(Transient Acantholytic Dermatosis)


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Grover's

disease.


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A Multiple

pruritic pink

papules on

the lowerchest (a

common

location),

some of

which are

crusted (B).

B Courtesy of

Jean L

Bolognia MD.


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Grover Disease


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(Transient Acantholytic Dermatosis)

Cause is unknown

The majority of patients are white males older than 40 years, with solar damage

Electron microscopy shows clumping of keratin filaments and loss of desmosomes

Immunohistologic staining reveals internalization and redistribution of desmosomalattachment plaques

Because GD shares several clinical, histologic, and ultrastructural features with DD,mutations in ATP2A2, the defective gene in DD, have been sought in patients withpersistent GD

No ATP2A2 mutation was identified in the skin (lesional and normal) or in leukocytes

in four patients, which indicates that GD is not allelic to DD. Thus, although DD andGD share striking clinical and histopathologic features, the mechanism underlyingacantholytic dyskeratosis in GD remains unknown.



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Grover Disease



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y y

Also called familial benign chronic pemphigus

Late-onset blistering dermatosis

First described by the Hailey brothers in 1939

Clinically: flaccid blisters and erosions are seen on the neckand in intertriginous areas, especially the axillae and groin;moist malodorous vegetations and fissures can develop

The two clinical variants are segmental type I and II

Histology: The major histologic finding is acantholysisthroughout the spinous layer, sometimes referred to as adilapidated brick wall



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An uncommon Autosomal Dominant Disorder

Mutations in the ATP2C1 gene (chromosome

3q21-24) result in dysfunction of a Golgi-

associated Ca2+ ATPase,

thus interfering with intracellular Ca2+

signaling

H il H il Di


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Loss-of-function mutations in the ATP2C1 gene

Encoding the

human secretory pathway calcium/manganese adenosine

triphosphatase (hSPCA1)

of the Golgi apparatus

which impair intracellular Ca2+ signaling.



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Hailey-Hailey DiseaseGolgi Apparatus

Mutations in the ATP2C1

human secretory pathway

Ca2+/Mn2+ ATPase

(hSPCA1)


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HaileyHailey

disease:

Erythematous

plaque in the

axilla with an

active border.


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Hailey

Haileydisease:

Erythematous

plaque with

erosions and

maceration of

the inguinal

canal andscrotum.


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HaileyHailey

disease:

Flaccid

vesicles and

erosions.


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HaileyHailey

disease.

Chronic

submammary

lesions with

erosions,

crusting andpainful

fissures.


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Histopathology of HaileyHailey disease: Crusting and suprabasilar as well as extensive acantholysis

throughout the epidermis. The latter has been likened to a

dilapidated brick wall.


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y y

Rare autosomal dominant Disease

Incidence suggested to be 1 in 50,000

Penetrance in adults is complete, but

expressivity is variable. Some forms present with

mild lesions resembling eczema, whereas other

forms are characterized by severe widespread

disease.

Hailey Hailey Disease


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The identification ofATP2A2 as the defectivegene in DD has guided the search for the genefor HHD. HHD had been linked to chromosomeregion 3q21-24, with no evidence for locus

heterogeneity.

Because HHD and DD are very similar bothclinically and histologically, the discovery of thecrucial role of SERCA2 in cell-to-cell adhesion

and differentiation of the epidermis raised thepossibility that defects in another calcium pumpcould underlie HHD.



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This led to the identification of a new gene in

the HHD region, ATP2C1, which encodes therelated

human secretory pathway Ca2+/Mn2+ ATPase

(hSPCA1),

as the defective gene in HHD



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The gene spans approximately 30 kb on 3q21 and comprises 28 exonsencoding a 4.5-kb transcript

The predicted protein is approximately 115 kd in size Alternative splicing ofATP2C1 primary transcripts in keratinocytes leads to four splice variants,ATP2C1a to ATP2C1d, which differ by different splicing of exon 27 and/or28

ATP2C1d is the largest variant, containing exons 27 and 28 in their entirety

ATP2C1 mRNA is ubiquitously expressed. It is highly expressed in humanepidermal keratinocytes and at variable levels in other human tissues

Behne and colleagues have shown that human keratinocyte SPCA1(Secretory Pathway Ca+2)localizes to the Golgi apparatus



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Hailey-Hailey Disease SPCA1 belongs to the SPCA (secretory pathway calcium ATPase)

class, which like PMCA (plasma membrane calcium ATPase)possesses only one of the two high-affinity Ca2+-binding sitespresent in SERCA

On the basis of amino acid sequence alignment, the structure of theSPCA family is thought to be similar to that of the SERCA family, butthe single Ca2+-binding site transports a single Ca2+ or Mn2+ ion

SPCA1 has been shown to transport both ions in the Golgi lumenand therefore plays a major role in cytosolic and intra-Golgi Ca2+and Mn2+ homeostasis

SPCA1 has the same apparent transmembrane organization andcontains all of the conserved domains found in P-type ion transportATPases. It exhibits 97 percent amino acid identity to the rat SPLACa2+ pump of the Golgi apparatus, 49 percent identity to the yeastsecretory pathway PMR1 pump, 37 percent identity to humanSERCA2, and 31 percent identity to human plasma membraneCa2+ ATPase PMCA2



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The studies have revealed that mutations did not cluster but were scatteredacross the ATP2C1 gene and show substantial allelic heterogeneity

Of these mutations, 20 percent are nonsense mutations and 30 percent areframeshift mutations, which indicates that at least 50 percent of ATP2C1mutations reported so far lead to PTCs

These mutations predict loss of expression or marked reduction of mutatedATP2C1 via nonsense-mediated mRNA decay, which further supports thepossibility that haploinsufficiency of ATP2C1 is a prevalent mechanism forthe dominant inheritance of HHD.

Nineteen percent of the mutations affect splice sites, but their effects onsplicing remains to be determined

Twenty-eight percent of the mutations are missense mutations, whereas theremaining 3 percent are in-frame mutations



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These mutations lead to amino acid changes inhighly conserved critical functional domains ofthe Golgi Ca2+ pumps in different species (rat,

yeast), and between SERCA and PMCA pumps

Some of these mutations occur at amino acidresidues, which by site-directed mutagenesishave been shown to abrogate SERCA1 function



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Comparison between genotype and phenotype,however, fails to detect any clear correlationbetween the nature of the mutation and theclinical features of HHD (age of onset, severity,

progression)

Extensive inter-familial and intra-familialvariation in clinical features has also been noted,

as well as variation between families sharing thesame mutation



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Majority of mutations result in

Protein instability

whereas others cause

Lack of Ca2+ and/or Mn2+ ion transport

through different alterations of the catalytic cycle ofthe SPCA1 pump



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Studies highlight the crucial role of specific residues in the binding ofCa2+ and Mn2+ to hSPCA1

They also further support the theory of haploinsufficiency as aprevalent mechanism for the dominant inheritance of HHD, whichsuggests that the level of functional hSPCA1 in epidermal cells is

critical

Functional studies in the yeast Saccharomyces cerevisiae alsosupport this interpretation

Yeast strains mutant for Pmr1, the yeast homolog of hSPCA1 (49

percent identity), are highly sensitive to cation chelators and Mn2+.These investigations showed that human HHD mutants exert nodominant negative effects on yeast expressing wild-type Pmr1,which is consistent with hSPCA1 haploinsufficiency as the basis forHHD.



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Previous studies of the yeast S. cerevisiae homolog Pmr1 haveshown that Pmr1-type pumps have a pleiotropic effect on Golgifunction. Null strains defective in Pmr1 show impaired proteolyticprocessing, incomplete N-linked and O-linked glycosylation, anddefective translocation of secreted proteins

In addition, these mutants are unable to degrade misfolded luminalER proteins

Mn2+ is an essential co-factor for a wide range of enzymes but istoxic when present at high concentrations

These studies have shown that PMR1 pumps are the principal routefor Mn2+ detoxification and are important in maintaining bothcytosolic and luminal Mn2+ homeostasis

Hailey-Hailey Disease In humans the SPCA pumps localize to the Golgi


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In humans, the SPCA pumps localize to the Golgicomplex, which they supply with Ca2+ and Mn2+required for the correct production, processing, and

maturation of membrane and secreted proteins

The Golgi apparatus is known to function as an IP3-sensitive Ca2+ store, like the ER.In addition, Ca2+ in thelumen of the Golgi apparatus controls protein trafficking,

cargo condensation, and precursor processing

Although the Golgi complex does not contain molecularchaperones, several Ca2+-binding proteins have beenidentified in the Golgi apparatus

Mn2+ in the Golgi apparatus is needed for N- and O-linked glycosylation of cellular proteins.



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In view of the ubiquitous expression of ATP2C1, it is not clearwhydefects in ATP2C1 result in a disease restricted to the skin, and why

the disorder is limited to certain areas of the skin

The mechanism by which mutant ATP2C1 cause acantholysis isalso unknown. HHD keratinocytes in culture display elevated restingcytosolic Ca2+ levels, abnormally low Golgi Ca2+ levels, andimpaired regulation of excess cytosolic Ca2+ in vitro



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Abnormal Ca2+ signaling in HHD keratinocytes correlates with decreasedprotein levels of ATP2C1. In vivo, clinically normal HHD contains lowerCa2+ stores and displays an abnormal Ca2+ gradient

Elevated cytosolic Ca2+ levels could influence gene expression or alterpost-translational modification of target proteins (activation of protein kinase

C)

Alternatively, low Ca2+ or Mn2+ concentrations in the Golgi lumen couldimpair post-translational modifications (proteolytic processing, glycosylation,trafficking, or sorting) of membrane (associated) proteins important inepidermal cell-to-cell adhesion, such as desmosomal components

This may impair desmosome formation and/orstability, leading to the acantholysis characteristic ofHHD.


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y y

It has been suggested that desmosomal

glycoproteins differ in their extent ofglycosylation among different cell types

Thus, the reason why clinical signs are restricted

to the epidermis could be related to a particularglycosylation state that leaves epidermal cellsmore prone to Ca2+ changes

It is also possible that non-cutaneous tissueshave compensatory mechanisms toovercome ATP2C1 dysfunction that aremissing in the skin



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The demonstration of loss of expression and/or function of mutantSPCA1 proteins identified in HHD suggests that epidermal cells aresensitive to levels of hSPCA1 (gene dosage), which may not be thecase for other tissues

This could explain why lesions are exacerbated by external factors(sun exposure, sweating, friction) that would place epidermal cellsunder stress and disrupt a subtle balance maintained in the steady

state

In support of this possibility, UVB irradiation and pro-inflammatorycytokines have been shown to downregulate ATP2C1 as well asATP2A2 mRNA expression

Finally, defective actin reorganization and a marked decrease incellular ATP have been reported in HHD keratinocytes in vitro,which suggests an additional mechanism in the pathogenesis ofHHD

Hailey-Hailey disease


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Hailey Hailey disease


Autosomal dominant

Flaccid blisters and erosions

Vegetating lesions at site of friction

Hx:

Suprabasilar acantholysis

Intraepidermal blister with villi

Dilapidated brick wall apearance

Rare dyskeratosis



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Hailey Hailey disease

Pathogenesis:.Autosomal dominant ATP2C1 gene Golgi-associated Ca ATPase

Altered intracellular Ca levels.

Low intra golgi Ca levels altered E-Cadherin processing.

High cytoplasmic Ca levels Activate protein kinase C

Phosphorylation of desmoplakin

Disruption of Desmosomes

References


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Fitzpatrick's Dermatology in General Medicine, 7th Edition

Klaus Wolff, Lowell A. Goldsmith, Stephen I. Katz, Barbara A. Gilchrest, Amy S.Paller, David J. Leffell

J. Dhitavat, R. Fairclough, A. Hovanian, S.M. Burge, Calcium pumps andkeratinocytes: lessons from Dariers disease and Hailey Hailey disease; Br JDermatol 2004: 150: 821-828.

W. Paschen, Dependence of vital cell function on endoplasmic reticulum calcium

levels: implications for the mechanisms underlying neuronal cell injury in differentpathological states; Cell calcium 2001, 29(1): 1-11.

J. Bolognia, J. Jorizzo, R. Rapini, Dermatology text book;

A. Kimyai-Asadi, et al, The molecular basis of hereditary palmoplantarkeratodermas CME article on Cutaneous Biology, J Am Acad Dermatol , September2002,47 (3): 327-339.

Medscape, emedicne.com, Google

Dr. Hee Young Park Slides

THANK YOU


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disorders of calcium

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