Biochemistry of skin

Jana Novotná

The role of the skin• System maintaining body homeostasis;

• Barrier

– to keep water and solutes in– against a range of noxious stress (UV irradiation, mechanical, chemical

and biological insults);– to prevent a desiccation and temperature balance;

• Contains sensory receptors for touch, temperature, pressure, pain, etc.

• Exteds to ~2 m2 in area, ~ 2.5 mm thick on average, constitutes 6%

of our total body weight (5 – 6 kg);• Protection to the UV radiation – absorbing pigmentation system;• Complex immuno-regulatory network protection;• Normal skin pH is somewhat acidic - the range of 4.2. to 5.6.

Human skin layers

• Mammalian skin is composed of two primary layers: – the epidermis, which provides waterproofing and serves as a

barrier to infection;– the dermis is responsible for the tensile strength of skin. Its main

functions is to regulate temperature and to supply the epidermis with nutrient-saturated blood. Much of the body's water supply is stored within the dermis.

Epidermis

• An external stratified, non-vasularized epithelium (75 – 150 m thick), continually keratinizing

- Stratum corneum – 15 – 30 sheet of nonviable, but biochemically active corneocytes (cornified - hardening, with keratin, lack cytoplasmic organells). Barrier against physical and chemical agents, reduces water loss

– Stratum granulosum – 3 – 5 sheet of non-dividing keratinocytes, producing keratino-hyalin

– Stratum spinosum – 8 – 10 sheet of keratinocytes with limited dividing capacity, Langerhan´s cells

– Stratum basale – maturing/aging keratinocytes, melanocytes, Merkel cells (receptor cells)

Keratins

• Keratinocytes contain filaments of the keratin intermediate filament (KIF) family (cytoskeleton)

• Hair, nails, horny layers of the skin – are formed from keratin cytoskeleton of dead cells.

• Two primary groups of keratins, the -keratins and the -keratins

-keratins occur in mammals, -keratins in birds, reptiles,

• Both form are right handed helical structure • 2 types

– type I – acidic keratins– type II – basic keratins– heterodimer – type I forming a coild coil with type II

Composition and structure of keratin

• Human skin contains ~ 20 genetically different keratins

• The most abundant amino acid are glycine and alanine, cysteine can account for up to 24%

• Long stretches -helix is interrupted by short non-helical segments

• Contact between 2 helices are formed by hydrophobic amino acid side chain on 1 edge of each helix

– two polypeptides form a dimeric colid coil

– protofilaments are formed from two antiparralel of head-to-tail associated coils

– protofilaments dimerize to form a protofibril and four of which form a microfibril

Composition and structure of keratin

• The cysteine residues in alpha-keratin affects its macromolecular structure and function.

• Hair has relatively few cysteine cross-links, whereas fingernails have many such cross-links.

• Intra- and intermolecular hydrogen bonds, disufid bridges occurre at all keratins.

• In cells, keratin type I forms pair with keratin type II • Different keratin types are expressed in different cell types and

different layers of epidermis:– cytoskeleton of epithelial cells - K14 (type I) & K5 (type II), K18 (type I)

& K8 (type II) – Basal layer – K13 (type I) & K4 (type II)– Spinus and granular layer – K10 (type I) & K1 (type II)– Stratum corneum – K3 (type I) & K12 (type II)– Hairs and nails – various other keratin pairs

Permanent waving is biochemical engineering

What happens to the keratin inside the hair during the permanent waving? The characteristic „stretchability“ of -keratins, and their numerous disulfide cross-linkage, are the basis of permanent waving.

The hair to be waved or curled is first bent around a form of appropriate shape.

A solution of a reducing agents (thiol or sulfhydryl groups, -SH) is applied with heat.

The reducing agent cleaves the cross-linkages by reducing each disulfide bond to form two Cys residues.

The moist heat breaks hydrogen bonds, causes the -helical structure of the polypeptide chain to uncoil.

After a time the reducing solution is removed, and an oxidizing agent is added to establish new disulfide bonds between pairs of Cys residues of adjacent polypeptide chains, but not the same pairs as before the treatment. The hair fibers now curl because the new disulfide cross-linkages.

The epidermal permeability barrier

• Barrier function in human epidermis depends on transglutaminase-mediated cross-linking of structural proteins and lipids („biological glues“).

• Transglutaminase reaction posttranslation modification of proteins, formation of covalent bond between a free NH3 group of protein- or peptide-bound lysine and the -carboxamide group of protein or peptide-bound glutamine free amine group.

• Proteins are than highly resistent to mechanical perturbation and proteolysis.

Fatty acids in epidermis

• The quality of the S. corneum barrier depends on the presence of equimolar concentration of ceramides, cholesterol and fatty acids.

• Changes in the concentration of any of these can affect barrier quality.

• Arachidonic acid and 20-carbon PUFA can be metabolized by either cyclooxygenase or lipoxygenase pathways → prostaglandins, hydroxyeicosatetraenoic acids.– phospholipids are starting point for the arachidonic acid pathway during

inflammation (allergic reaction)

• Some of these metabolites can interact with signaling system in proliferating and differentiating epidermal cells → modulation of protein kinase C, nuclear MAP-kinase cell apoptosis

Epidermal cell differentiation and turnover

• Basal keratinocytes transformation ~ 30 days to corneocytes.

• Damage cells are removed by normal squamation.

• Genetic damage - (UV-R) → trigger apoptosis (within hours) – „sunburn“ cells.– skin protection against UV-R concentrating of transferred melanin

over vulnerable keratocyte nucleus.

• Other insults can induce keratnocyte apoptosis – chemical, mechanical, immunological.

• The principal marker for keratinocyte/epidermal differentiation is expression of particular keratin pairs

Epidermal cell differentiation and turnover

• Proliferative basal keratinocytes express K5 and K14.

• In the early stages keratinocytes of maturation/differentiation switch to K1 and K10.

• The „pluri-potent“ stem cells for keratinocytes, sebaceous gland and epidermis rised from hair folicules.

• Ca2+ plays key role in epidermal differentiation - 4-fold increase of extracellular Ca2+ in S. corneum.

• Keratinocyte differentiation is regulated by hormones and vitamins - D3 and retinol from diet, thyroid hormone and steroid hormones.

• The skin has nuclear receptors for glucocorticoids, estrogen, androgen and progesterone.

Epidermal cell differentiation and turnover

• Important factors for keratocyte differentiation are Ca2+-dependent integrins – the receptors for the extracellular matrix fibronectin binding.

• Laminin and collagen IV and VII (basemen membrane components) – regulation of keratinocytes migration on basement membrane (very important during wound healing).

• Migrating keratinocytes produce many matrix metalloproteinases.

• Mature keratinocytes (in S. graulosum) contain protein-rich, keratohyalin granules and lipid-rich, lamellar granules.

• Lipids from lamellar granules form the sheets of the lipid permeability barrier of the epidermis.

Stratum corneum :anucleate, abundant keratin within cytoplasm, proteinaceous inner envelope, lipid outer envelope. Fillagrin – protein crosslinking keratin filaments, stored in keratohyalin granules.

Stratum granulosum:nucleated keratinocytes with keratohyalin granules. Keratinocytessynthesize flaggrin, release glycolipid lamellar product into intercellular space.

Stratum spinosus:polygonal keratinocytes, ovoid nucleus, abundant cytoplasm,engulfed melanosomes, many tonofibrils

Stratum basale:single layer attached to basement membrane, many hemidesmosomes

Melanocytes • Melanin-producing cells (S. basale) - precursor cell - melanoblast .

• Melanin is stored in the melanosomes .

• „Epidermal melanin unit“ - the anatomical relationship between keratinocytes and melanocytes.

• 1 melanocyte is in contact with ~ 40 keratinocytes

• Melanocytes extend arms to transfer melanosomes into the keratinocytes

Formation of melanosomes

• Melanosomes - elliptic membrane-bound organelles (melanin synthesis).

• Synthesis of matrix proteins and tyrosinase on the rough endoplasmic reticulum.

• Tyrosinase undergoes post translational modification in the form of glycosylation in the Golgi apparatus.

• Fusion of premelanosomes with coated vesicles containing tyrosinase - formation of the melanosome.

• Melanosome migrates into one of the dendrites of the melanocyte → transfer to a neighboring keratinocyte.

Melanin synthetic pathway• Melanin synthesis begins with

catalysation of the substrates L‐phenylalanine and L‐tyrosine to produce L‐DOPA via phenylalanine hydroxylase (PAH), tyrosinase and tyrosinase hydroxylase 1 (TH‐1).

• The pathways are then divided into eumelanogenesis or pheomelanogenesis.

• The other melanogenic enzymes are tyrosine related proteins - TRP‐2 (DCT) and TRP‐1 for eumelanogenesis.

• No specific enzymes have been found that are involved in pheomelanogenesis so far.

International Journal of Cosmetic ScienceVolume 33, Issue 3, pages 210-221, 25 JAN 2011 DOI: 10.1111/j.1468-2494.2010.00616.xhttp://onlinelibrary.wiley.com/doi/10.1111/j.1468-2494.2010.00616.x/full#f2

Melanins

• Melanins are polymorphous and multifunctional polymers of eumelanin, pheomelanin, mixed melanins (a combination of the two) and neuromelanin

• Mammalian cells produce black-brown eumelanin and yellow-redish pheomelanin

• Eumelanin - highly heterogenous polymer consisting of DHI and DHICA units in reduced or oxidized states.

• Pheomelanin - mainly sulfur-containing benzothiazine derivatives. • Neuromelanin - produced in dopaminergic neurons of substantia

nigra.• Melanin absorbs UV light at a wavelength of 280 - 320 nm• Both eumelanin and pheomelanin play important protective role in

binding to cations, anions, drugs, chemicals, etc.

Melanogenic regulatory proteins

The development of melanocytes is absolutely dependent on the action of the transcription factor, protein MITF (member of the family of basic helix–loop–helix leucine zipper microphthalmia-associated

transcription factors): – activation of regulatory network of transcription factors and

signalling pathways – control of the survival, proliferation and differentiation of

melanoblasts and melanocytes . – control of pigmentation via its transcriptional regulatory effect on

tyrosinase, TRP-1 and TRP-2. – MITF was shown to be a key transcription factor for a protein

important for melanosome transpor (Rab27A)

Paracrine melanogenic stimulators

• Melanotrophic hormones - proopiomelanocortin (POMC)-derived peptides (α-MSH, β-MSH, ACTH)

• POMC expression in keratinocytes is induced by UV -MSH, -MSH, or ACTH increases skin pigmentation predominantly in sun-exposed areas

• The POMC peptides exert its effect through a cyclic adenosine 3’,5’-monophosphate (cAMP)-dependent mechanism when binding to the Gs-protein-coupled receptor melanocortin receptor 1 (MC1R)

• Stimulation of specific Gs-protein-coupled receptors leads to the activation of adenylyl cyclase (AC).

• AC produces cAMP which consequently stimulates the melanogenic pathway the activation of protein kinase A (PKA) phosphorylation of enzymes, ion channels and several regulatory proteins eventually leading to a change in gene expression.

Factors involve in melanin production

• The melanin granules accumulate above the nuclei of keratinocytes and absorb harmful UV-R before it can reach the nucleus and damage the DNA.

• Quick responds of the melanocyte-keratinocyte complex to a wide range of environmental stimuli (paracrine and/or autocrine) - to UV-R, melanocyte-stimulating hormone (MSH), endothelins, growth factors, cytokines, etc.

• UV-R exposure → melanocytes increase their expression of proopiomelanocortin (POMC, the precursor of MSH) and its receptor melanocortin 1 receptor (MC1-R), TYR and TYRP1, protein kinase C (PKC), and other signaling factors.

• Fibroblasts (possibly other cells in skin) - produce cytokines, growth factors, and inflammatory mediators that can increase melanin production and/or stimulate melanin transfer to keratinocytes by melanocytes.

Other epidermal cells

• Langerhans cells - dendritic cells - arise from bone marrow early in embryonic development, occupy 2 - 8% of epidermis.– Important element of the immune system, interacting with T-cells – They are resided in suprabasal layer - attracted to keranocytes by E-

cadherin receptor– Their motion is regulated by specific integrin receptor and by α –TNF– They interact with the allergen in the stratum germinativum migration

to the lymphoid gland - then “teaching” of the T cells about the allergen– They specifically interact with T-lymphocytes and keratinocytes to

initiate host response to antigens (allergens)

• UV B stimulates synthesis and release of TNF- by keratinocytes which in turn modifies the behavior and morphology of Langerhans cells, decreases their total number.

AllergenLangehans

cell

T cell Activated T cell

cytokine

Other epidermal cells

• Merkel cells – receptor cells, location in stratum basale.

• They have synaptic contacts with somatosensory nerve endings (Merkel cell-neurite complex)

• associated with the sense of „light touch“ discrimination of shapes and textures.

Dermis • Responsible for the tensile strength of skin

• Main functions – regulation of temperature and to supply the epidermis with nutrient

• Much of the body's water supply is stored within the dermis

• Components:– connective tissue– hair follicles– sweat glands– sebaceous or oil glands– apocrine glands– lymph vessels– blood vessels

• The main cell type - fibroblast

Dermal proteins and extracellular matrix

• Collagen – about 90% of total dermal proteins – predominantly type I (85 – 90%), – type III (8 -11%), – minor type V (2 – 4%), (papillary dermis, matrix around vessels,

nerves), – type VI – associated with fibrils and interfibrillar spaces (responsible for

fine structure in early prenatal development of skin).

• Elastin, proteoglycans, glycoproteins, water and hyaluronic acid

Collagen, elastin, proteoglycans, glycoproteins structure - refer to lecture on Extracellular matrix

Skin appendages

• Skin plays in the body homeostasis, therefore is well-equiped with secretory (release of chemicals from cells for

physiological function) and excretory (elimination of weste

products of metabolism) capacity.– sweat glands [can be sweat secreted with strong odour

(apocrine) or with a faint odour (eccrine)].– sebaceous glands (secrete sebum onto hair follicle to oil the

hair).– hair follicle

Sweat glands

• 3 – 4 million eccrine sweat glands are in our skin – each producing water perspiration (serves principle to cool us) and maintain core temperature at 37.5oC.

• At maximum output the eccrine sweat glands can excrete as much as 3 l/hour, and heat loss is more than 18 kcal min-1.

• Humans utilize eccrine sweat glands as primary form of cooling.

• Apocrine sweat glands are larger, have different mechanism of secretion, and are limited to axila and perianal area.

Sweat secretion

• Eccrine gland activity is regulated via neural stimulation using sympathetic nerve fibers distributed around the gland.

• Neurotransmitter is acelylcholine• Sweating is controlled from hypothalamus (a center in the preoptic

and anterior regions), where thermosensitive neurons are located. • The stimulus for sweating:

– direct heating alone (39 to 46oC)– physiological sweating due to nerve reflex arise from sweat

center in brain cortex (emotional), hypothalamus (thermoregulation)

Eccrine sweat

• Contains mainly water (99.0 – 99.5%), electrolytes NaCl, K+ and HCO3

-, and other simple molecules - lactate, urea, ammonia, amino acids (serine ornithine, citruline, aspartic acid) and minerals.

• Mineral composition varies with the individual:– their acclimatisation to heat, – exercise and sweating, – the particular stress source (exercise, sauna, etc.), – the duration of sweating, and the composition of minerals in the

body

Apocrine sweat

• In lower mammals – secretion of pheromones (trigger sexual and territorial response).

• In humans – the significance of apocrine secretion of pheromones is not completely understood.

• Apocrine gland begin secreting at puberty.

• Apocrine duct exit to the surface via hair follicle.

• Apocrine sweat – more viscous, with milky consistency due to high content of fatty acids, cholesterol, squalene, triglycerides, androgens, ammonia, carbohydrates.

Mineral composition of sweat

sodium 0.9 g/l

potassium 0.2 g/l

calcium 15 mg/l

magnesium 1.3 mg/l

zinc 0.4 mg/l

copper 0.3 – 0.8 mg/l

iron 1 mg/l

chromium 0.1 mg/l

nickel 0.05 mg/l

lead 0.05 mg/l

Microelements

Sebaceous glands

• Glands secrete an oily/waxy matter, called sebum, to lubricate the skin and hair.

• Composition – 25% wax monoesters, 41% triglycerides, 16% free fatty acids, 1% squalene, small amount cholesterol esters and cholesterol

Skin metabolism

• Primary source for energy production in epidermis is glucose from circulation – diffuses into keratinocytes without effect of insulin. Large proportion of glucose is catabolized up lactate (even in presence of oxygen)

• citric acid cycle does operate in epidermis – explanation why this cycle is inefficient due to wide fluctuation of temperature and blood flow in skin.

• 20% of glucose is metabolized by pentose-phosphate pathway – production of NADPH and pentose for both FA synthesis and nucleic acids.

• Secondary source of energy - fatty acids derived from both epidermal stores and exogenous sources (when glucose flow is limited, then FA are metabolized).

Skin metabolism

• Glycogen – small amount under physiological conditions, however, elevation in all manner of injury of epidermis or during hair growth in follicle – explanation – energy when skin needs to be repaired or to use glucose immediately, most probably – disequilibrilium in metabolic processes

• Glucose is a substrate also for synthesis of lipids, polysaccharides and glycoproteins and nucleic acids

• GAG and proteoglycans – highly charged and attract water – forming gels (see GAG and proteoglycans in „Extracellular matrix“)

Skin metabolism

Lipid metabolism - components:

a) membranes,

b) major constituents of permeability barrier,

c) energy supply

Synthesis from both glucose catabolism and AA and circulating FA - lipogenesis is ongoing in all layers of epidermis - sebum synthesis – in sebaceous glands no accumulation of lipids from circulation; even in sexual maturation (higher synthesis of sebum) - increase of endogenous production and decrease of exogenous

Degradation - generally - lipases (yields in FA for neutral lipids – TG, sterol esters) – in outer layers of epidermis,

• specifically (e.g. formation of prostaglandins)

Skin immune system

• Skin not only provides immune protection for itself, but also for the whole body.

• Cell types containing battery of mediators of immune response– Langerhanse cells, monocytes, macrophages, mast cells

(cooperation with T-cells)• Cell types producing free radicals, anti-bacterial

peptides, cytokines chemokines, pro- and anti-inflammatory mediators– Neutrophils, eosinophils, basophils.

• B-cells secrete immunoglobulins (antibodies)

References

• D.J. Tobin. Biochemistry of skin – our brain on the outside. Chem. Soc. Rev. 2006; 35:52-67.

• G.E. Costin, V.J. Hearin. Human skin pigmentation: melanocytes modulate skin color in response to stress. The FASEB Journal 2007; 21: 976-994.

• J.M. Gillbro, M.J. Olsson. The melanogenesis and mechanism of skin-lightening agents – existing and new approaches. Int. J. Cosmetic Science 2011, 33: 210-221.

• I.F.S. Videira, D.F.L. Maura, S-.Magina: Mechanism regulating melanogenesis. An. Bras. Dermatol 2013, 88: 76-83.