**5. Matrix metalloproteinases: the ECM scissors**

Cells are required to maintain a specific collagen turnover. This delicate balance is crucial for remodeling activities while preventing surplus collagen deposition [33].

*Skin Aging: Implications of UV Radiation, Reactive Oxygen Species and Natural Antioxidants DOI: http://dx.doi.org/10.5772/intechopen.100102*

However, this balance is threatened by numerous agents. Pathways exist for intra as well as extracellular collagen degradation. One of the means of extracellular degradation involve MMP activity.

MMPs come under the Matrixin subfamily which belongs to Zinc metalloprotease family. They play a vital role in tissue reconstruction, cell migration, carcinogenesis, damage recovery and photoaging [34]. Since MMPs function to degrade ECM, their activities are heavily scrutinized by a cell [35]. MMP functioning can be inhibited by α-microglobulin as well as Tissue inhibitor of metalloproteinases (TIMPs). Further, many MMPs are produced as inactivated proenzymes [36]. UV triggered AP-1 expression tends to elevate MMP-1, MMP-3 and MMP-9 levels. Production of activated MMP-9 from human keratinocyte along with MMP-2 can digest type IV collagen found in the basement membrane. Further, they can also digest type V and VII collagen together with elastin [37]. MMP-1 can digest type I collagen which results in cell migration and damage recovery. MMP 14 (membrane bound MMP) can interact with TIMP-2 and pro-MMP-2 resulting in its activation [38]. Certain amount of collagen digestion also occurs due to MMP-8 released from neutrophiles [39]. Human macrophage elastase or MMP-12 acts on elastin fibers after which the degraded products accumulate in the epidermal layer resulting in solar elastosis and keratosis, the distinguishing features of photoaging [40].

### **6. Signaling routes: manipulation by UVR and ROS**

UV induced cell signaling is ligand independent. Therefore, how the cell perceives UVR is still not well defined. A particular study has revealed that Opsin-3 expressed by dermal fibroblast might help sense UVRs. Opsin comes under the GPCR family of receptors. Upon activation they lead to increase in intracellular calcium ion levels which further activate CAMKII. Activated CAMKII can in turn phosphorylate ERK, JNK, p38 and CREB. This event is ensued by rise of MMP-1, 2, 3 and 9 actions [41].

Initially studies conducted concentrated on activation via cytokine or growth factor receptor [42]. Epidermal growth factor receptor belongs to receptor tyrosine kinase (RTK). It is managed by protein tyrosine phosphatase (PTP). PTP bears a cysteine moiety in its active site which is originally employed for catalyzing a phosphohydrolase activity and ceasing the cell signaling cascade. This cysteine moiety can be targeted by ROS which would activate RTK for a prolonged period till ROS are dealt by cellular antioxidants [43]. Activation of receptors leads to recruitment of adaptor proteins for example Src homology-2 [SH2] domains and Src homology 3 domain [SH3]. SH2 interacts with phosphorylated tyrosine residue of the active RTK. SH3 in turn interacts with a phosphorylated proline residue on other targeted proteins. Through this they lead to stimulation of other downstream molecules [44]. Ras near the cell membrane is a guanine nucleotide binding protein that shuttles between GTP/ GDP bound form. The GTP bound form interacts with and activates Raf [45]. Raf sequentially activates MAP kinase kinase/MEK via phosphorylation. This is ensued by origination of active ERK [46]. These MAPKs can interact with several proteins and activate them. Interactions are mostly formed with conserved landing sites on the substrate. Jun D has two domains, a D domain and DEF domain. D domain is bound and phosphorylated by JNK under stress while DEF domain is phosphorylated by ERK which is activated by growth factors binding to EGFR receptor [47]. In presence of UVR and/or hydrogen peroxide, overexpression of c-Fos/Jun D or Jun D alone could prevent apoptosis by lowering Caspase-3 levels. Expression of c-Fos plus Jun D

also activates AP-1. Transcription factor AP-1 is a dimer of basic region leucine zipper protein which may constitute of Jun [c-Jun, Jun B and Jun D] and Fos [c-Fos, Fos B, Fra-1 and Fra-2] subfamilies in the cell. This complex is further joined by proteins from ATF and JDP subfamilies [48, 49]. AP-1 and TGFβ/Smad signaling cascade control procollagen synthesis. TGFβ are cytokines that allow pro-collagen formation by binding to their receptors, TβRI and TβRII. Upon meeting of TGFβ with their receptors, Smad 2 and 3 are activated which complex with Smad 4 to form a heteromeric molecule. This complex enters nucleus and helps in transcription of TGFβ regulated genes. In contrast, AP-1 inhibits procollagen formation coupled by destruction of existing collagen fibers by upregulating MMPs [50]. Interestingly, effects of AP-1 are subject to participating dimer proteins. Theoretically, AP-1 can appear as 18 different homo and hetero dimer and this can influence its activity. It is hypothesized that this could be as of differential DNA binding along with different transcriptional activity. Furthermore, the differentiation status of cell also influences the properties of different AP-1 transcription factor forming proteins [51].

Besides this pathway, other cascades such as JNK and p38 MAPK Pathway are also stimulated. c-Jun NH2 terminal kinases [JNK] are activated upon receiving stress signals such as cytokines, ROS, and UVR. A specific stimulus activates the MAP3Ks. The MAP3K catalyze phosphorylation of MAP2K. MAP2K has two isoforms, MKK4 and MKK7. Active MKK4 and MKK7 interact and generate active JNK. Some of its targets comprise of nuclear proteins like AP-1, Elk-1 and c-Jun. p38 MAPK is stimulated under stress [52, 53]. p38 MAPK is switched on by upstream lying MKK3 and MKK6 kinases. If JNK pathway is initially active then MKK4 of the JNK pathway can activate p38 MAPK. Apart from this, p38 can also undergo auto-phosphorylation. p38 MAPK is noteworthy as of its high interactivity with multiple nuclear proteins along with protein kinases [54]. Although many investigations involving UVR and cell fate have taken place, there are still certain gaps. A molecular switch exits between p38 and p53 proteins. If damage due to UV radiation is less, the cell opts to repair and survive. Under this situation p38 and p53 are activated separately. p53 stimulates p21waf1/CIP which stops cell cycle from advancing ahead by regulating Cdc25A/B. This leads to arrest in G1/S and G2/M phase. But if cellular damage is too high then p38 phosphorylates p53 resulting in active p53 that initiates apoptosis [55].

Recently it was shown that p38 MAPK can stimulate HIF when keratinocytes are irradiated with UVR. p38 activates HIF-1 which in turn activates Noxa. Active Noxa catalyzes degradation of Mcl-1. Mcl-1 controls differentiation and survival of keratinocytes. Therefore, its degradation leads to apoptosis. This cascade might be affected by ROS as ROS can stimulate Ask/p38 MAPK pathway. Again, HIF-1 is also responsive towards redox status in the cell [56]. Secretion of cytokines like IL-8 can stimulate the three MAPK pathways. Its activation can turn on ATF-2 nuclear protein which induces elevated secretion of MMP-3 in fibroblast [57].

Since UVR amplifies ROS generation, a prime pathway activated under such conditions is the NFκB pathway. NFκB pathway activation leads to transcription of numerous genes coupled with immunological and inflammatory responses. NFκB family constitutes of 5 proteins that is p65 (Rel A), Rel B, c-Rel, p50 (NFκB 1) and p52 (NFκB 2). Stimulation of suitable receptors upon ligand binding activates IKK (IκB kinase). IKK constitutes of IKKα, IKKβ and IKKγ. The IKK molecule phosphorylates IκBα. After phosphorylation, IκBα is poly-ubiquitinylated at lysine moieties which marks it for degradation by proteasome. The resulting active NFκB is then transported to nucleus for transcription of specific genes. Some of the prime signaling events are illustrated in **Figure 1** [58, 59].

*Skin Aging: Implications of UV Radiation, Reactive Oxygen Species and Natural Antioxidants DOI: http://dx.doi.org/10.5772/intechopen.100102*

**Figure 1.** *Schematic diagram showing the important signaling pathways involved in Photoaging.*
