Hair regeneration and follicle development is a possible thing in human adults thanks to the Wnt/beta-catenin pathway. One important factor that drives the Wnt/beta-catenin pathway is the reliance on the zinc finger protein 5, which is type CXXC. This type works as a counter controller and thus enhances hair regrowth and enhances hair follicle neogenesis through its association with Disheveled. The first type of hair loss is called the AndroGenic Alopecia (AGA) attributed to the androgens found in genetically susceptible males and females ( Kim & Garza, 2017) . Hair follicles depict specific characteristics in AGA such as increased 5-alpha-reductase enzyme activity and high levels of dihydrotestosterone (DHT). The DHT and testosterone have varied activity regarding their action on the androgen receptors. DHT binds strongly to the androgen receptors as opposed to the testosterone. This results in the activation of genes responsible for the alteration of terminal follicles into miniaturized follicles as seen in AGA ( Premanand & Rajkumari, 2018) . The FDA has only approved Finasteride, topical minoxidil, and 5-alpha-reductase as the viable treatments of AGA.
Figure 1: Mechanism of Wnt secretion
Notably, the Wnt/beta-pathway is very important especially in the development and regeneration of hair follicle in the adults. Moreover, understanding how the Wnt/beta-catenin enhances this regeneration and follicle development helps in the management of AGA ( JoséLeiróset al., 2017) . It does so by salvaging the DHT-induced suppression of the hair follicle differentiation of the stem cells that lead to the miniaturization of the hair follicles. The activation of the Wnt pathways modulates cell generation, survival, and behavior in both young and old. The pathway starts in the nonexistence of Wnt ( MacDonald, Tamai, & He, 2009) . The cytoplasmic β-catenin creates a compound with Axin, GSK3, CK1, and APC which is phosphorylated by CK1 being blue subsequently by yellow GSK3 ( SookChoi, 2013) . The phosphorylated β-catenin is known as E3 ubiquitin ligase β- Trcp . This aims at β-catenin for proteasomal reduction. Groucho (HDAC) represents the Wnt target genes. With Wnt target ligand, a receptor compound is created between Fz and LRP5/6 ( JoséLeiróset al., 2017) . The application of dvl by Fz results in both LRP5/ 6phosphorylation and axin disrupting Axin -mediated degradation of β-catenin. As a result, it is collected in the nucleus from where they form the site and acts as activators for TCF to trigger Wnt reactive genetic factor ( MacDonald, Tamai, & He, 2009) . The pathway is as shown in the figure below
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The figure below summarizes the Wnt/beta-catenin motioning pathway during cell differentiation, growth, proliferation, and survival.
The instigation Wnt/beta channel can come about because of treatments with inhibitors such as the glycogen synthase kinase 3beta. Other activators of the pathway such as the valproic acid (VPA) and hair-stimulating complex are responsible for the hair follicle cell proliferation. The CXXC5 is a small protein that has individual characteristics such as possession of the CXXC-type zinc-finger domain, suppresses the Wnt/beta-catenin pathway, and is a target gene for the Wilms Tumor 1 ( JoséLeiróset al., 2017) . The experiment utilized quantitative analysis using immunohistochemically-staining methods. The protein programmed by this inheritable factor is a retinoid inducible nuclear protein, which contains zinc finger motif type CXXC. The prearranged protein is tangled in myelopoiesis, and is required for DNA mutilation induced p53 stimulation, controls the division of C2C12 myoblasts to form myocytes, and adversely control cutaneous wound curing. It has been found that many transcript variations programs. Whereas the role of the CXXC5 in inhibiting the Wnt/beta-pathway has been proven, its function in the therapeutic site for the AGA is unclear yet ( Lee et al., 2017) .
Wound-induced hair follicle neogenesis method can also be used in handling hair loss issues. The neogenesis (WIHN) works by regenerating follicles through first, the activation of Wnt/beta-catenin pathway, in the present action of wounding ( JoséLeiróset al., 2017) . However, wounding results in a de novo regeneration of the hair whose efficiency is relatively low. As such, there is a necessity to have an activator whose function is to increase the efficiency of regeneration. Probably, the efficiency of the regeneration process could be achieved using peptide drugs and small molecules, something that is yet to be approved. Therefore, the research sought to analyze the status of CXXC5 on both the haired and the bald patients' scalps suffering from AGA ( Kim & Garza, 2017) . Secondly , the study will also aim at assessing the effect of the competitor peptide that disrupts the CXXC5-Dvl interaction critical to hair regrowth and the expression of WIHN and ALP both in vitro and vivo ( CXXC5 CXXC finger protein 5) .
One of the critical results found out from the study is the fact that CXXC5 is overly expressed in miniaturized hair follicles both in the balding scalps of humans and catagen follicles found in the skins of mice. Investigating the clinical effects of Wnt/beta-catenin and CXXC5 in AGA required monitoring of the levels of beta-catenin and CXXC5 from tow subjects, one haired, and the other bald both of whom have AGA ( Lee et al., 2017) . From the experiment, excessive expression of localized beta-catenin was seen in hair matrix cells of the subject with a haired bald scalp but the contrary was observed in the balded scalp. Normal hair follicle on the hand showed little to no expression of CXXC5. However, the expression was excessive in miniaturized follicles of individuals having a bald scalp. Regarding ALP, its expression was highest at the apex of the cells located in the dermal papilla of the haired scalps. However, ALP levels were almost undetectable in the scalps of bald individuals ( Kim & Garza, 2017) . Other elements that showed high expression in the bald scalp include the keratin 15 (hair follicle stem marker), CD34, and CD200 both of which are progenitor cell markers.
Patients with AGA also show an inverse relationship between the patterns of beta-catenin and CXXC5. In evaluating the essence of CXXC5 in hair growth in the mice, the beta-catenin and CXXC5 were monitored at the adult cycle. The methods used included the western blot and immunohistochemical, which depicted that the beta-catenin levels were highly expressive especially in the anagen phase whereas the CXXC5 peaked during a phase known as the anagen-catagen transition ( Lee et al., 2017) . The second postulation from the study was that CXXC5 had a role in the suppression of the activity of ALP and attenuated the proliferation of human hair follicle because of the interaction with Dvl-1. The human hair follicle dermal papilla cells (HFDPCs) plays a critical role in helping to determine the function of CXXC5 in modulating the activity of ALP and cell proliferation. It is apparent that CXXC5 has an inverse relationship with ALP , beta-catenin, and PCNA. Immunocytochemical analyses backed with ALP staining showed that CXXC5 diminished the levels of both ALP and beta-catenin.
Another critical conclusion drawn from the experiment was that losing CXXC5 was an essential step in promoting the regeneration of hair both in the wounded and normal skins. Moreover, treatment with VPA resulted in the phenotypic inducement of CXXC5-deficient mice. Evidently, the mice treated with VPA shown an increasing acceleration of hair regrowth ( Lee et al., 2017) . In contrast , on the application of a combination treatment involving Wnt3a and PTD-DBM synergistically led to the expression of ALP, beta-catenin, HFDPCs, and PCNA. The Wnt/beta-catenin pathway can easily be deactivated by disintegrating the CXXC5-Dvl interaction where a peptide is used. The peptide has three properties including a protein transduction domain, a linker, and a lysine ( Premanand & Rajkumari, 2018) .
It is also important to determine the role of CXXC5 as the controller of Wnt/b-catenin pathway that follows negative feedback pathway. It is also a critical thing to note that the co-treatment seen with the use of VPA and PTD-DBM leads to the regrowth of hair ( Kim & Garza, 2017) . Moreover, the co-treatment results in ALP inducement, Beta-catenin, and PCNA expression in mice. In proving these assertions, the experiment involved shaving the dorsal skin area of the mice and subsequently treating the shaved area with VPA and PTD-DBM. Strong fluorescence, especially in the epidermis and stratum corneum, was detected on treatment with PTD-DBM within thirty minutes after treatment.
However, when the treatment involved VPA and PTD-DBM the effects observed was markedly different. The combination treatment led to an accelerated regrowth of hair much more than a single treatment with any drug of choice. The combined treatment also led to other benefits such as an increased hair weight and an improved hair follicle number. Dermal thickness was another improvement seen from the combined treatment. Further benefits seen with the combined treatment as confirmed with immunohistochemical analyses included increased expression of PCNA and beta-catenin. However, one thing that remained constant irrespective of the treatment method employed was the skin architecture. The proper maintenance of the keratin expression plays a role in body protection. PCNA, ALP, and beta-catenin all showed a synergic increase after a period of combined treatment ( SookChoi, 2013) . However, the keratin 14 levels remained unchanged in all the treatment types. It is imperative to note that the arrangement of the keratin 14 mainly determines the skin architecture. Another critical revelation that became apparent from this experiment was the treatment with the VPA lead to an interaction of CXXCD-Dvl. However, the CXXC5-Dvl interaction was markedly affected by the treatment with PTD-DBM. Immunohistochemical and quantitative analyses both revealed that the combined treatment with VPA and PTD-DBM resulted in the inducement of the PCNA and beta-catenin in the hair bulb and the epidermis of the skin of the mice.
Co-treatment with VPA and PTD-DBM is associated with two effects such as the enhancement of WIHN and the marked inducement of beta-catenin and fgf9 expression in the wounds of the mice. While investigating the combination treatment of WIHN, the experiment utilized wounds of one centimeter in diameter inflicted on the backs of mice and then treatment made using VPA and PTD-DBM. The results showed that the combined treatment critically enhanced WIHN in the wounds of the mice. The combined treatment first led to the marked increase in both fgf9 and beta-catenin, which subsequently led to the increased levels of WIHN. Dermal and epidermal proliferation both increased in the combined treatment as seen using a nalyses by both western blot and immunohistochemistry. Another element that increased with the combined treatment includes k eratin17 neogenic f ollicles. VPA and PTD-DBM co-treatment also resulted in the expression PCNA and beta-catenin in the epidermis and dermis as viewed through the quantitative a nalyses enabled by the immunohistochemical staining. It is also important to appreciate the positive feedback loop exhibited between fgf9 and Wnt pathways during the manifestation of WIHN ( JoséLeiróset al., 2017) . As such, the experiment sought to address this by monitoring the levels of beta-catenin and fgf9 in the wound dermis. The observation led to the conclusion that there was a simultaneous inducement of both fgf9 and beta-catenin in the mice wounds that had been treated in combination with VPA and PTD-DBM.
Staining with ALP was critical in showing the co-treatment with VPA and PTD-DBM markedly increased WIHN. It also led to the emergence of new unpigmented hairs at the core of the re-epithelialized wounds at around 25 days from the day of wounding. Moreover, the interaction seen in CXXC5-Dvl is a possible strategy that could be used in the development of novel drugs that lead to hair follicle neogenesis. The expression of the CXXC5 also differs in the dermal papilla under different circumstances ( Kim & Garza, 2017) . The dermal papilla of a bald hair follicle showed a high expression of CXXC5 while a slight expression was seen in a dermal papilla of a hair follicle that is normal. The expression of the CXXC5 began in the anagen-catagen transition and advanced to its peak during the catagen phase ( Lee et al., 2017) . The anagen-catagen transition is characterized by proteins involved in the inhibition of the hair growth such as fibroblast growth factor 5 and prostaglandin D2. Therefore, the CXXC influences the inhibition of hair growth as seen in both human and mice.
The Wnt/β-catenin pathway activation results in the inducement of the CXXC5. However, it leads to the inhibition of this pathway through a negative feedback mechanism in the papilla cells of humans and mice. The strange expression of CXXC5 in the hair follicles could be caused by the scarce expression of the beta-catenin. It is also vital to note that DKK1, which is a secreted Wnt antagonist, comes because of upregulation by the action of Wnt3a in human dermal papilla cells ( Premanand & Rajkumari, 2018) . In patients suffering from AGA , DKK1 is upregulated in the balding scalp as compared to how the same happens in the haired scalp of the same patients. Therefore, the study found out that the treatment target for patients with hair loss is the CXXC5. However, there is a gap in knowledge that first needs to be resolved with regards to the mechanism involved in the upregulation of CXXC5 in patie0nts having AGA ( Lee et al., 2017) . It is also imperative to note that WIHN is a vital tool in the treatment of hair loss. After the closure of the wound, the Dkk1 is transiently inducted leading to the abrogation of WIHN. Lastly, the over-expression of a component known as the Wnt7a in the epidermal area increases WIHN.
The Wnt/β-catenin pathway regulates some cell processes through regulating the absorption of transcriptional controller β-catenin. The proteasomal deprivation of β- catenin is interceded by 2-β-transducing recurrences, which contain protein (beta-Trcp) paralogues HOS and FWD-1. This is a functional interchangeable and is considered as a function which is in excess. Wnt/β-catenin signaling control both HOS and FWD1 although counter-clockwise directions, thus creating interconnected negative and positive response twists. In this control loop, the practical importance of reverse control of HOS and FWD1 by Wnt/ beta-catenin gesturing in the presence of inactive accomplishments in a proteasomal deficiency of beta-catenin is an unsolved issue. Through research, it was discovered that in remote conditions the signaling conditions are majorly influenced by HOS feedback arising from high absorption of HOS. Further, the changing of forward regulation of FWD1 by other pathways reduces the effect of the HOS reaction. Reverse regulation of HOS and FWD1 expression by Wnt/β-catenin signaling permits application of FWD1 reaction as a counter-mechanism compared to abnormal pathway initiation due to condensed HOS concentration.
Most importantly, fgf9, which results from alpha theta T cells, leads to the activation of Wnt in the wound fibroblasts leading to the inducement of WIHN. It is critical to note that hair regeneration after wounding does not apply in humans as happens in the mice. The same does not happen in humans because they have low levels of sequestered alpha theta T-cells. Therefore, they lack the healthy hair regeneration after a period of wounding. The efficiency of the WIHN is partly increased by testing activators such as Fgf and Wnt pathways during the process of wound healing. The co-treatment discussed earlier that comprises of VPA and PTD-DBM also increases the chances of wound healing ( Kim & Garza, 2017) . The combined treatment also facilitates a strong potential for asserting WIHN, which is instrumental in the initiation of pathways including Wnt and FGF pathways. The hair regeneration, production of pigment, and protein signaling occur because of various pathways working in synergy. Some of them include the stem cell factor, Wnt/beta-catenin, and bone-morphogenic protein pathways. Such pathways are collectively referred to as the multiple signaling pathways. The pathway is needed in the differentiation into melanocytes in the skin’s surface.
When the melanocytes lose the vital component- beta-catenin- the result is the formation of unpigmented hair. Consequently, when the Wnt/beta-catenin pathway undergoes an ectopic activation in the melanocytes, the result is the emergence of premature hair graying. The mice skin used in the experimentation did not show any signs of abnormalities concerning hair pigmentation after combined treatment with VPA and PTD-DBM. Hair regeneration and the treatment of hair loss can be solved using a combined treatment that targets the initiation of Wnt/beta-catenin pathway and blocking CXXC5-Dvl interaction using a peptide ( Kim & Garza, 2017) . The success of the experiment required the use of specific materials and methods in determining the therapeutic possibilities of different pathways in developing a treatment for hair loss and enhancing hair regeneration. The first material that was used efficiently was the human tissue sample . The importance of the human tissue sample stemmed from the fact that the experiment wanted to investigate the effect the impact of CXXC5 on the development of baldness. The males were used in this experiment because they are most likely to be affected by baldness and hair loss.
The hair sample included those from haired and bald scalped individuals. The total number of men involved in the study was five all of whom suffered from the AGA. The hair was obtained using a 5mm biopsy punch. However, it is critical to appreciate that the experiment followed the highest ethical standards; this case they correlated with the Declaration of the Helsinki Principles. All patients used in the experiment were required to give informed consent before engaging in the research study.
The tissue specimen obtained from the men were fixed in 10% formalin overnight and were kept in a phosphate buffered saline for purposes of immunochemical studies after that. Adherence to the ethical guidelines was an essential step in performing this experiment to reduce the chances of backlash from various quarters given that the study is invasive and might involve pain in the AGA patients. The mice were also an essential part of the experiment. However, a particular type of mice was required to analyze the propensity of the action of CXXC5 in determining hair behavior among the mice. The CXXC5 heterozygous mice were manipulated through intercrossing for four generations to achieve littermate wild-type mice that could be suitable for the said experiment. In using the mice, ethical guidelines were also adhered to mainly because the study involved wounding them and determining the action of various components of hair growth in that region.
Some significant conclusions can be drawn from the experiment. However, it is vital to recall the central question that prompted the research in the first place is to determine the suitability of Wnt/beta-catenin pathway and manipulation of CXXC5 especially in the treatment of baldness and the regeneration of hair. One of the important conclusions made is that it is the primary reason for the loss of hair and increased baldness in both humans and animals. As per the experiment, it is highly expressive in miniaturized hair follicles in people having balding scalps. Therefore, the therapeutic strategies in the treatment of AGA should focus on inhibiting the action of CXXC5. Another important conclusion relates to the role of the combined treatment with VPA and PTD-DBM is vital in the hair regeneration and prevention of hair loss. Through the experiments with the mice, it is evident that the combined treatment is better placed to solve the hair loss situation due to its activity in activating ALP, beta-catenin, HFDPC, and PCNA.
Overall, the study sought to find a solution to AGA disease found in humans and other animals. The disease characterizes itself through loss of hair leading to the baldness of the skull. However, with the knowledge that Wnt/beta-catenin pathway is responsible for the development of hair follicles, valuable research can target this pathway to evaluate several factors that could inhibit it. Therefore, the experiment utilized bit human, and mice cells in the course of the experiment and many trials are made utilizing various components of the skin and hair. From the results of the experiment, it is apparent that the action of CXXC5 is inhibitory to the pathway hence leading o hair loss in an individual. Therefore, therapeutic targeting of AGA should target the CXXC5. Moreover, the results suggest that combined treatment is also important in the pursuit of desirable results. Finally, the ethical considerations are also important especially regarding the question of test subjects whether human or animal are to be used. Certain gaps in knowledge, however, exist such as the rationale of CXXR5 in inhibiting the Wnt/beta-catenin pathway hence calling for further research.
References
CXXC5 CXXC finger protein 5 [ Homo sapiens (human) ]. https://www.ncbi.nlm.nih.gov/gene/51523
JoséLeirós, G. et al., (2017). Androgens modify Wnt agonists/antagonists expression balance in dermal papilla cells preventing hair follicle stem cell differentiation in androgenetic alopecia. Molecular and Cellular Endocrinology, 439 : 26-34. https://doi.org/10.1016/j.mce.2016.10.018
Kim, D. & Garza, L.A. (2017).The Negative Regulator CXXC5: Making WNT Look a Little Less Dishevelled. Journal of Investigative Dermatology 137(11): 2248-2250. https://doi.org/10.1016/j.jid.2017.07.826
Lee, S-H., Seo, S. H., Lee, D. H., Pi, L-Q., Lee, W-S., & Choi, K-H. (2017). Targeting of CXXC5 by a Competing Peptide Stimulates Hair Regrowth and Wound-Induced Hair Neogenesis. Journal of Investigative Dermatology, 137 (11): 2260-2269. https://doi.org/10.1016/j.jid.2017.04.038
MacDonald, B. T., Tamai, K., & He, X. (2009). Wnt/β-catenin signaling: components, mechanisms, and diseases. Dev Cell. 2009 Jul; 17(1): 9–26. doi: 10.1016/j.devcel.2009.06.016
Premanand, A. & Rajkumari, R. (2018). Androgen modulation of Wnt/β-catenin signaling in androgenetic alopecia. Archives of Dermatological Research. pp 1–9. doi: 10.1007/s00403-018-1826-8
SookChoi, Y. (2013). Distinct Functions for Wnt/β-Catenin in Hair Follicle Stem Cell Proliferation and Survival and Interfollicular Epidermal Homeostasis. C ell Stem Cell, 13 (6): 720-733. https://doi.org/10.1016/j.stem.2013.10.003
