The Property Assessed Clean Energy (PACE) Program is an innovative financing approach to meeting environmental goals. PACE financing is structured as an assessment to the property and paid along with the property tax bill. In addition to the direct environmental benefits, it also yields co-benefits of enhanced economic output and employment. This paper estimates the economic impacts of PACE in California by one of its major financing companies. These impacts include direct spending on structural improvements, reduction in spending on centralized power and water services, reallocation of spending from energy and water bills savings, and solar investment tax credits, among others. It also includes general equilibrium effects of these various factors. Our results indicate PACE financing yields sizable economic benefits. At the same time, the increased economic activity results in increased energy and water use that partially offsets some of the direct environmental gains. Furthermore, PACE has been subject to criticism because it gives financing companies the first lien on mortgages and because of anecdotal examples of some customers being lured by unscrupulous contractors. The direct environmental benefits and economic co-benefits of PACE should be factored into the policy debate over whether the Program should be further expanded or regulated. •Property Assessed Clean Energy provides innovative financing for environmental goals.•Ygrene Energy Fund has financed over 32,000 CA projects since 2013 for $760 million.•Reduced net electricity use by 3.53 million MWh and natural gas use by 2.02 bcf in CA.•Increased the NPV of GDP by $661 million and employment by 9,774 person-years in CA.•Economic co-benefits should be considered in discussions of regulation and expansion. Established by state statutes and enabled by local governments, PACE financing, unlike traditional alternatives, is an assessment to the property and not the property owner. Only certain types of improvements are eligible to be financed through PACE, and all PACE programs are required to provide public benefits in the host state. PACE provides one hundred percent of the cost of qualified structural improvements, which the property owner repays annually or semi-annually through a special assessment added to the property tax bill.co-benefits directly and indirectly. These benefits are more complex than those measured in ordinary economic impact analysis associated with typical economic stimuli such as the opening of a new factory or mine. Such stimuli, as well as PACE financing, all generate direct impacts on site and multiplier or broader general equilibrium impacts throughout the rest of the economy. However, many conventional stimuli provide products primarily for export and do not interact much with the host economy beyond a limited number of supply chain and wage/salary income increases and spending. PACE improvements, on the other hand, provide energy and water cost savings that stimulate spending on consumer goods and enhance business profits. At the same time, the efficiency improvements reduce the production of conventional energy and centralized water services and affect their prices. Hazard mitigation improvements help prevent the need to dip into savings to repair damaged structures and compensate for business interruption.The direct benefits of PACE financing are rather straightforward—reduction in energy and water use, reduction in pollutants associated with this use, and reduction in hazard vulnerability. However, the financing also generates economic This study performs a regional economic impact analysis of PACE financing by one of the leading firms in the market—Ygrene Energy Fund, Inc. Since 2013, and through the end of July 2018, Ygrene has provided more than $1.16 billion to finance over 54,500 property improvement projects in over 500 cities and counties in California, Florida, and Missouri . We estiThis paper fills an important void in the literature on the total regional economic, environmental, and resource use impacts of PACE financing. Most of the analyses of PACE financing have dealt with its strengths and limitations  that are not quantified in this study, add to the “business case”, “household benefits” and “overall societal well-being” of PACE financing.Between its start in 2013 through July 2018, Ygrene financed 31,867 residential properties and 646 commercial properties in California.33.1Estimating the economic impacts of the PACE Program involves a number of considerations. It includes several positive stimulus factors, as well as some partially offsetting ones. Some of these impacts relate to the buildings in which improvements are made, while others relate to direct and indirect impacts off-site. Many of the benefits of the PACE Program pertain to improving the environment and are complex to assess because of the absence of market prices associated with them.A set of positive stimulus factors that directly affect the economy stems from the purchase of materials and equipment, as well as the labor involved in their installation, for PACE improvements. Examples include energy-efficient water heaters, solar panels, windows, caulking material, and wages paid to workers with various skill levels. The demand for the various inputs  into the PACE improvements directly increase the economic activities of several sectors that produce these inputs, which leads to further upstream and downstream interindustry multiplier effects. The demand also leads to the payment of wages/salaries to workers and returns to capital to business owners, which they subsequently spend, setting off further rounds of multiplier effects. Additional positive stimuli emanate from the operation and maintenance of PACE improvements throughout their useful life.Another set of positive stimuli is associated with the savings from improved efficiency in the use of energy and water. Homeowners then use the major portion of these savings to purchase more of the typical basket of consumer goods, thereby generating additional economic activity. Business owners could use the savings for several purposes, including expanding production, lowering prices, or increasing wages and profits.Similar savings stem from mitigation efforts that reduce vulnerability to disasters. Here, however, the assessment of spending is more complicated, because, in the absence of these improvements, property owners would spend money anyway to repair and reconstruct damaged facilities. On the surface this could be an equivalent amount to spending on the improvement and would appear to be a wash in terms of direct economic impacts as measured by standard economic indicators such as gross output or employment. However, implementing disaster mitigation improves the level of well-being of the property owners by reducing their risk of loss of property or income, while repair and reconstruction simply returns well-being to the original level. The well-being of homeowners and businesses are measured in this report in terms of their expenditures on hazard mitigation.A set of direct substitution effects also needs to be taken into account, which offsets the aforementioned positive stimuli to a lesser, equal, or greater extent. Reduced spending on energy and water results in a decrease in the economic activity that produces them. The net effect is likely to be positive within most states, however, since the majority of the positive stimulus spending will increase the activity of in-state producers, while the inputs to displaced production are top-heavy with goods imported from other states .Additional positive stimuli stem from interest and fees paid to Ygrene and from fees paid to state and local governments for operating costs, including permitting and inspection. Fees paid to governments at various levels increase their revenues and are likely to be spent on government provision of goods and services within a state, thus generating an additional direct stimulus. Payments to Ygrene increase its business activity, and hence wages and profits, with the former subsequently spent primarily within the state.Yet another stimulus associated with PACE financing stems from the 30% solar energy federal investment tax credit (ITC). This payment essentially increases the disposable income of households and increases consumer expenditures on goods and services, on the one hand, and increases business profits, or possibly lowers prices, on the other. The credit is usually received by the property owners between 4 and 16 months after the installation of the solar energy system. We assume that the households will spend 50% of the credit dollars in the year of the installation, and 50% in the following year. Moreover, since the solar ITC is a federal tax incentive, we assume there will be no reduction in federal government spending in California or increase in other taxes in the state as an offset effect.Finally, we summarize some additional benefits and downsides of PACE financing, the estimation of which are beyond the scope of this study. There is evidence that the financed improvements increase the value of the home or business, in part because of the lower energy and water costs, as well as reduced vulnerability to hazards. A complex issue of the analysis is that the PACE Program provides assessments rather than outright grants. Since these assessments need to be repaid, this results in a diversion of expenditures from other goods and services by consumers or from production activities by businesses. Thus, with respect to the spending from and repayment of the assessments, there is a significant stimulus in the year(s) that the improvement is implemented, but a negative stimulus to the economy spread over the period during which the assessments are repaid. This repayment can come equivalently from prior savings or assets of the borrowers or from savings on the utility bills and insurance premiums that the improvement provides, or some combination of both.Even the initial use of the assessments may involve some offsetting factors depending on the origin of the capital base of a PACE Program lender. The assessment of the PACE Program is being done at the local level within a state, so, if the capitalization of the lender comes from funds from out-of-state, then there is an equivalent positive stimulus from all the assessments. However, if the capital comes from within the state, we must evaluate whether it displaces the use of these investment funds for other purposes. If so, this displacement effect would have to be taken into account, and the ensuing direct and indirect impacts would have to be subtracted from the bottom-line impacts. The difficulty in addressing this component is that firms that invest in PACE securitizations are active in global markets. While many firms have headquarters in the US, some in states where PACE programs operate, we do not have access to their geographical investment portfolio. Thus, estimating where funds originated would be extremely difficult if not impossible. Given the relatively small amount of PACE financing as a proportion of total global capital market funds available, we believe that an assumption of zero in-state investment displacement is reasonable.3.2A major set of direct impacts stems from reduced water use, reduced energy use, and reduced greenhouse gas emissions. Unfortunately, directly observable market prices do not exist for pollutants, so we will apply some measures in the literature of the damages they cause, where the benefits of PACE assessments represent these avoided losses. For example, we use the findings of a recent National Academies of Science report on the social cost of carbon 3; howeveIn the case of the benefits of decreased energy use and water use, we encounter two complexities. First, we could apply the value of these resources, but this would overstate the benefits because we have already counted them in the consumer savings. At the same time, simply using the values of these resources at market prices omits the consumer and producer surplus components and causes some underestimation. Hence, we simply present the amounts of energy and water savings in physical units.3.3As noted above, the spending on property improvements that are made possible by Ygrene financed local government assessments apply to the site of their application, or what is termed direct economic impacts. However, the estimation of the total, or macroeconomic, impacts includes the ripple, or multiplier, effects of the various increased or decreased spending streams, as well as the interaction of demand and supply in numerous markets. For example, the increased demand for high-efficiency water heaters directly stimulates the demand for inputs into their production, such as fabricated metals, insulation material, and labor. The process continues through a chain-reaction of indirect impacts, as more fabricated metal and insulation production stimulates demand for more of their inputs, and as the producers of these inputs demand more inputs, and so on.The many types of linkages in the economy and macroeconomic impacts are extensive and cannot be traced by a simple set of calculations. They require the use of a sophisticated model that reflects the major structural features of an economy, the workings of its markets, and the interactions between them. In this study, we used the Regional Economic Models, Inc. (REMI), Policy Insight Plus (PI+) Model . This is4A summary of the data used in this study is presented in Appendix Ygrene Energy Fund, Inc. provided us with extensive data on its financing from 2013 through July 2018. The data covered major characteristics of the individual assessments, including major assessment characteristics , financing characteristics , and characteristics of the property .The authors then mapped improvement expenditures to the 160 economic sectors in the REMI Model to calculate the indirect effects. This began by dividing these expenditures between sectors that produced/supplied the property improvement materials/equipment and installation of the equipment or retrofit of the structure.4Data on energy and water savings were obtained from the “Ygrene Proprietary Impact Metrics Model” developed by Ygrene. This impact model is used to estimate the energy savings, water savings, natural gas savings, renewable energy generation, utility bill savings, and the associated greenhouse gas emissions reductions. The impact model was based on a 12-month data-set of Ygrene funded projects between July 2017 and June 2018. For each residential and commercial property in the data set, Baseline Energy Consumption by end-use  and Baseline Water Consumption by end-use  were determined to form the foundation upon which improvement level savings are estimated.After the energy  and water savings quantities were estimated at the improvement level, state-level energy and water rates for residential and commercial sectors and emissions factors were applied to these savings quantities to generate associated utility bill savings and greenhouse gas reductions.All energy, water, utility bill, and greenhouse gas saving estimates were then divided by the total contract amount  within each of the improvement categories to derive the energy, water, utility bill, and greenhouse gas savings factors per thousand contract dollars by improvement type, state, and property type. These factors are then applied to the entire Ygrene portfolio based on the contract amounts for each improvement type, state, and property type to estimate annual savings.To estimate lifetime savings, annual factors are applied for the useful life of each improvement type. When calculating lifetime utility bill savings, it is assumed that the prices of electricity, natural gas, and water will increase or decrease at the average annual historical growth rates over the past 6 years in each state . AdditioMitigation Saves 2 Study  for the earthquake threats for each improvement type. The BCRs were obtained from results calculated as part of the  2 Study . Note thNote also that due to the uncertainty surrounding some of the data, below we perform sensitivity tests around best estimates of data inputs into the analysis.55.1The implementation of Ygrene PACE improvements is estimated to result in substantial direct benefits from energy consumption reductions and water conservation. In California, the energy/water efficiency and renewable energy PACE projects are estimated to lead to electricity consumption reductions of 3.63 million megawatt hours (MWh), natural gas consumption reductions of 2.86 billion cubic feet (bcf), and water savings of 2.36 billion gallons over the entire useful life of the improvements. 75.2State-specific emissions factors were applied to the energy saving quantities to calculate the associated greenhouse gas reductions.5.3Mitigation Saves 2 Study  of the PACE Program. It is the most widely used macroeconometric model to analyze the economic impact of energy and climate policies in the U.S. The REMI Model has evolved over the course of more than 30 years of refinement  model, i.e., it divides the economy into 160 sectors, and thereby depicts important distinctions among them.The REMI model is able to analyze the quantity interactions between sectors (ordinary multiplier effects) but with refinements for price changes not found in I–O models. That is, the Model incorporates the responses of producers and consumers to price signals and the changes in other market and regulatory conditions, and captures the substitution effects and other price-quantity interactions. The REMI Model also brings into play features of labor and capital markets, as well as trade with other states or countries, including changes in competitiveness. The labor market in the REMI model is linked to a demographic module of population migration. It also includes input substitution between labor and other factors of production, market supply and demand, wage rate determination, and economic geography considerations of labor accessibility of individual industries.The econometric feature of the REMI Model refers to two considerations. The first is that the model is based on inferential statistical estimation of key parameters based on pooled time series and cross-regional (panel) data. This gives the Model an additional capability of being able to extrapolate the future course of the economy, a capability that most other types of economic impact models usually lack.The version of the REMI Model used includes two geographical regions: California and rest of U.S. The model is established based on U.S. and California historical data through 2016. However, in this study we primarily use the California region to simulate the economic impacts of PACE investment in that state.Before undertaking any economic simulations, the estimates of the direct costs and savings of the PACE projects are translated to REMI PI + model inputs. This step involves the selection of appropriate economic activity and policy levers in the Model. 7In the Base Case, we utilize our best estimates of key input variables. For example, these include the average BCRs for earthquake improvements and average insurance savings. They also include our assumption that property owners will repay their financing by displacing their other spending by an equivalent amount. Also, we do not include offset effects of reduced electricity and natural gas demand for the investment displacement in the Base Case analyses. Various adjustments of all of these inputs and assumptions are made in the sensitivity tests below.7.1The results indicate that, during the up-front investment period (2013–2018) of the Ygrene PACE financed property improvements, an average annual increase of GSP of $134.7 million and employment of 1,305 jobs10We also performed decomposition analyses for the GSP and employment impacts to evaluate how the various economic factors affect the aggregate macroeconomic results.Note that the positive impacts of the non-market production of electricity can be added to REMI Model results for Gross Output  to obtain an “Augmented Gross Output” result. Analogous “Augmented GDP” and “Augmented Personal Income” can be estimated by applying value-added or personal income coefficient of the electric utility sector to the “Augmented Gross Output” result. Note, however, that the non-market electricity production does not generate any employment.7.2As our focus is on estimating the macroeconomic co-benefits of PACE financing, we only provide a brief summary of the direct benefits: reductions in water and energy consumption, in GHG emissions, and in losses from natural disasters. It is important to note that these reductions are brought about by financing that basically pays for itself in terms of cost savings on utility bills and avoided costs from the need to repair homes and commercial buildings. The reader is referred to the first partition of Total GDP impacts  are $661.4 million for California. The employment impacts are 9,774 person-year jobs. The largest contributor to these impacts emanate from Energy Cost Savings, followed by the net impacts of Ygrene Financing, Interest Payments and Other Program Fees minus Annual Repayment of this Financing.Total AGDP impacts  are nearly $850 million. The value of Non-Market Electricity Production becomes the third largest contributor to these impacts.7.3In evaluating the contribution of PACE financing to reduce GHG emissions, natural resource use and hazard reduction, it is also important to consider any offsetting effects. Ironically, the same economic stimuli that are the focus of this report have the potential to engender this “offset” effect, in that increases in economic activity generate additional GHGs and utilize more water, natural gas and electricity. The increased amounts emanate from direct effects in the production of inputs into building improvements and the spending of workers who receive income from producing and installing the improvements. They also emanate from the supply-chain effects of production and further consumer spending of additional income generated, as well as consumer spending from energy and water bill savings and reduced costs of natural disasters.11Note that the offset effects for GHGs and electricity are a minimal 12.5% and 2.7%, respectively. However, the offset is a significant 29.4% for natural gas and much more than offsets the direct reductions in the case of water. Note, however, the offset effect represents only 7 ten-thousandths of a percent of baseline water use in the state.Overall, as with regard to most policy improvements, there are some offsetting effects in relation to stated objectives and/or negative side-effects in other domains. Policymakers must weigh these trade-offs in evaluating the policies. In the case of Ygrene financing, the net environmental gains are still positive in three of the four cases. For the remaining case of net increases in water use, it should be noted that those offsets are larger than the savings because of the small percentage of water conservation projects in the state.8We perform several sensitivity tests to analyze how the changes in some key assumptions would affect the macroeconomic impact analysis results of the PACE financing.8.1We first perform a sensitivity test on our assumption that people need to reduce other purchases to repay PACE financing on a dollar-for-dollar basis. It is possible that they will dip into savings, so as not to fully displace other spending. We therefore performed a sensitivity test that reduces the direct offset by 10%. The GDP and employment impacts are increased by 11.4% and 13.0%, respectively, indicating that the Base Case results are not very sensitive to this assumption.8.2We also include the dampening impacts from the decreased demand for electricity and natural gas from the energy supply sectors. These impacts are simulated as decreases in exogenous final demand from the Electricity Generation sector and Oil and Gas Extraction sector in the REMI simulations.The primary reason for excluding this offset in the Base Case is that California law (under Senate Bill 350) requires the state to increase the share of electricity derived from renewable sources from 33% to 50% and to double the efficiency of existing buildings. Additionally, the California Energy Commission (CEC) established the Existing Buildings Energy Efficiency Action Plan to increase energy efficiency in residential, commercial, and government buildings. All of these policies and regulations are entirely independent of PACE, and most of them pre-dated the establishment of PACE programs in these states. Thus, PACE only serves to assist in achieving efficiency and renewable targets that are already mandated by state law and Public Utility Commission regulations. However, it is possible renewable energy and efficiency targets will more than be met by responses to market conditions, in which case PACE displacements of electricity and natural gas would represent an overage and justify including their value in the reduction of electricity and natural gas production. In our sensitivity test, we have thus assumed a 50% offset.Under this offset assumption, the Base Case GDP impacts are reduced from $661.4 million to $482.7 million. Thus, the estimated economic impacts are somewhat sensitive to the inclusion of the offsetting effects of energy demand reduction. This is because over 92% of the Ygrene PACE improvement investment in California is energy efficiency upgrades and renewable energy installations. The esimates for California are most sensitive to this assumption, resulting in a reduction of Base Case GDP Impacts by 27%.8.3A question arises as to the extent to which the various improvements simulated here would have taken place in the absence of Ygrene financing, i.e., whether the impacts are truly additive. Estimates by 99.1PACE financing, like many other government or government-sanctioned programs, has been justified not only by the direct benefits it would generate but also by various co-benefits, one of them being their potential to stimulate the economy. It cannot be taken for granted that positive net economic benefits are forthcoming for every new program, and a careful study of economic impacts, such as that presented above, is warranted. It is then up to policymakers in their program evaluation to determine how much weight to give to these co-benefits. One approach is to juxtapose them along with the direct benefits, of course, to any downsides of the policy/program.PACE financing has not come without criticism. The most significant opposition comes from the Federal Housing Finance Agency (FHFA) concerning the PACE assessment's super lien status, opposition shared by the Mortgage Bankers Association and the National Association of Realtors. PACE financing is voluntarily applied to the property as a special assessment added to the property tax bill, and as such, like all property taxes, is senior to any first mortgage, and thus in the event of foreclosure, only the portion of the PACE assessments that are due at that point in time are paid out before the mortgage. In 2010, the FHFA, as conservator of government sponsored enterprises (GSEs) Fannie Mae and Freddie Mac following the 2008 financial crisis, instituted policy that Fannie and Freddie would not purchase or refinance a mortgage with a PACE assessment. This policy was based on the objection that PACE assessments added risk to Fannie and Freddie's assets by displacing them as prime mortgage holders in the event of property owner default and foreclosure. It is important to note, however, that because PACE is a voluntary special assessment, and cannot be fully accelerated by the taxing jurisdiction, the PACE administrator, or any entity other than the property owner, only the annual PACE assessments in arrears would be due in the event of foreclosure. Thus, the risk to Fannie and Freddie as a result of PACE is not the entire PACE obligation itself, but only the annual assessments past due. Typically, PACE assessments cannot exceed 10–15% of the fair market value of the property, and the combined loan-to-value ratio of PACE and the underlying mortgage cannot exceed 90–100% depending on the state's PACE statute.In December of 2017, the Federal Housing Agency (FHA) issued Mortgagee Letter 2017–18, joining FHFA's opposition to PACE, instituting a policy that it would not insure any new mortgages with a PACE assessment, reversing its previous position put in place in 2016 that it did insure mortgages with PACE assessments. The FHA cited similar reasons as FHFA for its objection to PACE. However, the California Alternative Energy and Advanced Transportation Financing Authority (CAEATFA) established a State-funded loan loss reserve program in 2010 that provided first mortgage holders the ability to recover any losses as a result of residential PACE in the event of foreclosure. Of course, one solution, should default issues accelerate , would b13FHFA and, to a lesser extent, FHA policy opposition toward PACE is one of the most significant barriers to the expansion of residential PACE enabling legislation and broader PACE market expansion. Initial PACE legislation was established in California in 2008. Soon after California, many other states enacted PACE legislation. Today, thirty-six states, as well as Washington DC, have enacted PACE legislation, twenty of which, and Washington DC, have active commercial PACE programs. Yet, only three states – California, Florida, and Missouri – have active residential and commercial PACE programs operating simultaneously.Additionally, there has been some criticism from individuals and consumer protection organizations arguing that PACE financing lacks regulation and needs stricter guard rails to protect consumers from potential abuse from unprofessional contractors and misunderstandings about how PACE works. California has addressed many of these concerns through recent legislationCalifornia's recent legislation, FHFA and FHA policy toward PACE, and broader banking and realtor opposition to PACE have, and will continue to have, significant influence on the future of the national, state, and local PACE markets. As state legislators, federal regulators, and policy makers consider how to approach PACE policy, we intend that our research will play a role in how that policy takes shape. For example, future PACE policy could have a significant impact on whether California will meet its GHG emission reduction goals under Assembly Bill 32 passed in 2006, its goal to double the energy efficiency of existing buildings by 2050 under Senate Bill 350 passed in 2015, and, most recently, its goal to achieve 100% renewable energy by 2045 under Senate Bill 100 passed in 2018.As this research shows, PACE programs can have significant positive impacts on GDP, job creation, energy and water conservation, natural hazard mitigation, and greenhouse gas emission reduction, among others. These positive impacts exceed any negative impacts measured to date.9.2The Property Assessed Clean Energy (PACE) Program has broadened significantly in recent years and now also includes financing for saving water and for reducing vulnerability to disasters for both residential and commercial properties. It is able to achieve its direct societal objectives while providing financial gains to those receiving financing and implementing the building improvements. It does so by providing financing that is beneficial to the recipients by saving money on utility bills and avoiding having to pay for building repairs or reconstruction following an earthquake. Moreover, it represents an equitable alternative collateralization of the financing, in part because the property value is not a criterion for qualifying nor is a credit score, thereby making it available to some who could not otherwise secure financing through more conventional lending-related instruments.We have estimated the direct benefits of PACE financing by one of its major administrators, Ygrene Energy Fund, Inc. More uniquely, we have also estimated the broader macroeconomic co-benefits of this financing in terms of impacts on market-based GDP and augmented GDP that takes into account the non-market effects. The analysis was performed in the context of an Economic Consequence Analysis that took into consideration the numerous positive and negative stimuli associated with PACE financing. The macro impacts were estimated with the use of the REMI Policy Insight Plus Model, the most widely used macroeconometric model at the state and local levels in the US.Total Augmented GDP (AGDP) impacts, which include the non-market value of solar electricity production, social cost of carbon, and avoided disaster relocation cost, are nearly $850 million for California. Employment impacts are the same as the regular employment impacts noted above, since new jobs are not directly associated with any of these direct environmental and hazard reduction benefits.Sensitivity tests indicate our results are robust to changes in major assumptions relating to displacement effects of repaying PACE financing, offsetting effects of energy demand reduction, alternative estimates of insurance savings, and the relative additivity of Ygrene financing. Overall, the PACE financing provided by Ygrene, and even more so if we included all PACE financing, generates sizable net positive impacts on the economies of the major states of operation.co-benefits, which are very positive on net. These co-benefits are thus also likely to be of considerable interest to policy-makers evaluating future decisions about accommodating and even facilitating PACE financing in addition to the broader public policy goals of achieving federal, state, and local energy, environmental, and disaster mitigation goals.PACE is a public policy tool designed to leverage private capital to help homeowners and business owners overcome barriers to implementing building improvements and to provide broader public benefits. PACE financing has successfully reduced air pollution emissions and resource use but has faced criticism by competitors and some analysts because it is not traditional in terms of collateral and because of some marketing and quality control issues of a small percentage of independent contractors that implement the various improvements. The industry addresses this through compliance guidelines for each PACE program. In addition to the more straightforward direct benefits of PACE financing, this paper provides estimates of the several economic PACE is a financing mechanism by which broader and more equitable access to property improvements in energy efficiency, water conservation, renewable energy, and hazard mitigation is made possible. This research shows that PACE has had a positive net impact on the environment and the economy in one of the major states in which it is enabled.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


TP63), progenitor (SOX9), quiescence (CEBPD), and proliferation  genes and the upregulation of genes for differentiation  in treated- compared to non-treated samples. The pathway activation was shown by AXIN2 upregulation and enhanced levels of accumulated β-catenin. Immunocytochemistry and Western blot confirmed the findings for most of the above-mentioned markers. The Wnt/β-catenin signaling profile demonstrated an upregulation of WNT1, WNT3, WNT5A, WNT6, and WNT11 gene expression and a downregulation for WNT7A and DKK1 in the treated samples. No significant differences were found for WNT2, WNT16B, WIF1, and DKK2 gene expression. Overall, our results demonstrate that activation of Wnt/β-catenin signaling in ex vivo expanded hLESCs governs the cells towards differentiation and reduces proliferation and stem cell maintenance capability.Human limbal epithelial stem cells (hLESCs) continuously replenish lost or damaged human corneal epithelial cells. The percentage of stem/progenitor cells in autologous ex vivo expanded tissue is essential for the long-term success of transplantation in patients with limbal epithelial stem cell deficiency. However, the molecular processes governing the stemness and differentiation state of hLESCs remain uncertain. Therefore, we sought to explore the impact of canonical Wnt/β-catenin signaling activation on hLESCs by treating ex vivo expanded hLESC cultures with GSK-3 inhibitor LY2090314. Real-time qRT-PCR and microarray data reveal the downregulation of stemness ( The corneal epithelium is a dynamic, transparent, multilayered structure composed of non-keratinized human corneal epithelial cells (hCECs) that acts as a physical, chemical, and immune barrier to the cornea and the rest of the eye . The supReduced number and/or function of hLESCs, so-called limbal stem cell deficiency (LSCD), leads to the inability to maintain an intact corneal epithelium . The culWnt/β-catenin signaling, an evolutionary conserved molecular pathway involved in stem cell fate determination ,23, is hAs mentioned above, the ex vivo expansion and cultivation of hLESCs from limbal biopsies and, thereafter, transplantation (CLET) is one of the most commonly used methods for the treatment of LSCD. However, most of the previous studies have focused their research on Wnt/β-catenin activation on hLESCs isolated as single-cell cultures, and the previous data regarding the Wnt/β-catenin signaling in ex vivo expanded hLESCs from limbal biopsies are scarce ,29,30,31In the present study, we aimed to test the effect of the small molecule LY2090314, a GSK-3 inhibitor and Wnt/β-catenin activator, on the cell fate of hLESCs expanded from healthy limbal biopsies cultivated ex vivo. In addition, we searched for particular Wnt signaling ligands and pathway molecules upon Wnt/β-signaling activation in hLESCs.All tissue collection complied with the Guidelines of the Helsinki Declaration. The laboratory procedures and tissue harvesting were approved by the Regional Committee for Medical and Health Research Ethics, Norway (No 2017/418). Unless stated otherwise, all reagents were purchased from Merck .2+ and Ca2+ free Hank’s Balanced Salt Solution  for 10 min at 37 °C, followed by washing in Fetal Bovine Serum . The limbal biopsies were plated with the epithelial side down on six-well TC-Treated Multiple Well Plates . The biopsies were submerged in complex medium (COM) containing DMEM/F12 , 100 U/mL penicillin, and 100 μg/mL streptomycin (P4333), 2.5 μg/mL Amphotericin B (A2942), 5% FBS (F2442), 2 ng/mL epidermal growth factor (E9644), 5 μg/mL insulin, 5 ng/mL sodium selenite and 5 μg/mL transferrin (l1884), 30 ng/mL cholera toxin A subunit from Vibrio cholerae (C8180), 0.5% dimethyl sulfoxide , and 15 μM hydrocortisone (H0888). Once the biopsies attached to the well, they were completely immersed in COM and incubated at 37 °C, 5% CO2, and 95% air for the following nine days. The culture medium was changed every 2–3 days.The preparation of limbal biopsies and in vitro expansion of hLESCs was performed according to the standard protocol . Corneosn = 3–6) were used for further analysis. In addition, DMSO reciprocal to the DMSO concentration in cultures treated with 10 nM LY2090314 was added to individual wells as controls. The DMSO dilution of 10 nM LY2090314 concentration in COM medium was 1:10,000. Images from primary hLECS cultures were acquired using a light microscope .The hLESCs proliferated and expanded from the limbal biopsies in COM on the six-well plates for nine days. Thereafter, the COM was removed and replaced by COM containing 1 nM, 2 nM, 5 nM, and 10 nM of LY2090314, when applicable. One to three wells per condition per donor  was used; LDH release was measured from the collected medium according to the manufacturer’s instructions. The colorimetric values of three replicates were monitored at a wavelength of 490 nm on a Victor3 multilabel plate reader. Once the primary hLESCs growing out of the limbal biopsies reached confluence, they were treated with 0.25% Trypsin-EDTA (T3924) and seeded on 24-well plates  at 6 × 10n = 5) containing: (1) untreated, control hLESCs cultured in COM and (2) treated hLESCs cultured in COM containing 5 nM LY2090314 harvested for nine days. The medium was removed and replaced by a fresh medium every three days. RNA isolation for Real-Time qRT-PCR analysis was performed from the paired individual wells of the six-well plate from five human donors (−ΔΔCt comparative method was used to interpret the fold change (FC) in gene expression and standardize results to the endogenous reference gene, 18S values. Total RNA was isolated from the non-treated and treated hLESCs using 1–3 individual wells of six-well plates for each condition. RNA isolation was carried out using an RNeasy Micro Kit  and quantified via spectrophotometry . Reverse transcription (RT) was performed using a high-capacity cDNA Reverse Transcription Kit  with 1 μg total RNA per 20 μL RT reaction, followed by cDNA dilution to 2 ng/μL using 180 μL RNase/DNase free water . The StepOneplus Real-Time qRT-PCR (Applied Biosystems) and TaqMan Gene expression assays, including predesigned primers/probes  was used for the analyses. 2 transformed signal intensities was applied for normalization (http://bip.weizmann.ac.il/toolbox/overview/Partek_Users_Guide.pdf) accessed 12 September 2020. Gene transcripts with maximal signal values of less than 5  across all arrays were removed to filter out low and non-expressed genes. The differentially expressed transcripts between groups “treated vs. control” were identified using two-way ANOVA as implemented in Partek Genomics Suite. The results are expressed as FC. Genes with FC ≥ 1.5 and a p-value < 0.05 were regarded as significantly regulated.We subjected 150 ng of total RNA to the GeneChip WT PLUS Reagent Kit, following the manufacturer’s protocol for whole-genome gene expression analysis . The labeled and fragmented single-stranded cDNAs were hybridized to GeneChip Clariom S Human . The arrays were washed and stained using an FS-450 fluidics station . The signal intensities were detected using a Hewlett Packard Gene Array Scanner 3000 7G . The scanned images were processed using AGCC (AffymetrixGeneChip Command Console) software. For statistical analysis of the gene expression, the Affymetrix CEL files (containing probe intensities) were imported into Partek Genomics Suite software . A robust microarray analysis (RMA) yielding normalized loghttps://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE234403, accessed on 29 April 2023).The Affymetrix gene expression data were deposited in NCBI’s Gene Expression Omnibus  and are ® goat anti-rabbit IgG  for samples stained with Ki-67, p63α and SOX9, and Alexa Fluor® 488 donkey anti-rabbit IgG  for antibody stained with Cx43. Next, the nuclear staining was performed with Hoechst 33342 staining  for 15 min at room temperature. Finally, the samples were washed 3 times with DPBS for 10 min and mounted  onto slides. The fluorescence was recorded using a ZEISS Axio Imager M1 fluorescence microscope . Nuclear antibody positivity  was counted by three independent individuals. The primary cultures growing out from limbal biopsies were washed with Dulbecco’s Phosphate-Buffered Saline  and fixed with 4% formaldehyde for 15 min at room temperature, then washed again 2 times with DPBS and permeabilized with 0.5% Triton X-100 (93443) for 15 min. After that, the cells were blocked with 5% BSA in 0.1% Triton X-100/DPBS for 30 min. The primary antibody was dissolved in 0.5% BSA/0.1% Triton X-100/DPBS and incubated for 3 h at room temperature. The following primary antibodies were used: proliferation marker Ki-67 , progenitor markers: tumor protein p63 alpha , SRY-Box Transcription Factor 9 , and a differentiation marker: connexin-43 . After incubation, the slides were washed 3 times with DPBS for 10 min. Incubation was continued with the appropriate animal secondary antibody type: Cy3Equal protein concentrations (30 μg) previously measured using albumin standards  were separated by size using electrophoresis on 4–15% Criterion TGX Gels . The proteins were then further transferred from the gels to PVDF membranes by semidry electrotransfer. The membranes were blocked with 5% dry milk or 3% BSA in Tris-Buffered Saline/TBS)/0.1% Tween buffer  for 1 h at room temperature. The 1× TBS/0.1% Tween solution was also used as a washing buffer and a diluent for the antibodies. Thereafter, the membranes were incubated with anti-Cx43 polyclonal rabbit  or anti-β-catenin  overnight at 4 °C. The membranes were washed three times for 5 min, followed by 1 h of incubation at room temperature with the same-origin horseradish peroxidase-linked whole secondary antibody . After washing, the protein–antibody complexes were detected using the enhanced chemiluminescent assay after adding the substrate . The band intensities given by Cx43 were normalized to Vinculin using a monoclonal anti-Vinculin antibody produced in mouse , and the band intensities given by β-catenin were normalized to β-actin using anti-β-actin antibody produced in mouse  and the appropriate secondary antibody. g at 22 °C. After cell counting, the cells were plated on coverslips in 12-well TC-Treated Multiple-Well Plates . The coverslips were previously coated with poly-L-lysine (P4707) for 1 h. Thereafter, in the different paired individual wells of the control and treated cells from three individual donors, 10 μM EdU  was added for 1 day, from day 1 to day 3, from day 4 to day 6, and from day 6 to day 9, so that the EdU could incorporate into the DNA during cell replication during these time intervals. Thereafter, the cultured cells were washed 3 times with PBS containing 3% BSA and fixed with 4% formaldehyde for 20 min, then washed again with 3% BSA in PBS and permeabilized with 0.5% Triton X-100 in PBS for 20 min. Ultimately, the cells were stained with a reaction cocktail that was made according to the manufacturer’s instructions for 30 min protected from light. Then, the nuclei were stained with 5 µg/mL Hoechst 33342 solution. The coverslips with cells were mounted on slides and fluorescent images were taken accordingly. Quantification of the EdU-positive cells was carried out the same as for previously mentioned markers. The primary cell cultures were washed with DPBS and trypsinized in 0.25% Trypsin-EDTA for 10 min, blocked with FBS dissolved in DMEM/F-12, and centrifuged for 10 min on 302× U test, and p values < 0.05 were considered to be statistically significant.Descriptive statistical methods, mean ± SEM, were used to describe the gene and protein expression values from the available replicates of the same donors and group of donors. The statistical analyses were performed using Prism 8.3.0 . After the data were tested for normal distribution (Shapiro–Wilk test), the significant differences between the two groups were determined using a parametric unpaired two-sample t-test or nonparametric Mann–Whitney The primary hLESC cultures treated with LY2090314 appeared to have a changed morphology, cell size enlargement, and a more pronounced nucleus and nucleoli when compared to controls . The celp < 0.05), whereas this measurement was not higher in hLESCs upon treatment with 2 nM concentration and DMSO control. However, no statistical difference was found after nine days of treatment in the LDH release between treated and control hLESCs , KRT3 , and pathway control gene AXIN2 , whereas significance was not found in cytokeratin 12  gene expression (TP63 and CCAT/enhancer binding protein delta (CEBPD), and those regulating limbal basal/progenitor cell maintenance, SOX9 as well as proliferation, MKI67, and proliferation cell nuclear antigen (PCNA) were significantly downregulated in treated hLESC cultures   B. WNT1, WNT3, WNT5A, WNT6, and WNT11 genes , whereas significant downregulation was found for WNT7A and DKK1 . No statistically significant difference in the mRNA levels and hence gene expression could be found for WNT2, WNT16B, WIF1, and DKK2   . p-value (p < 0.05) and multiple testing correction (FDR < 0.05). Nine genes were expressed at higher FC levels  and five genes were expressed at lower FC levels  in the treated hLESC cultures. p-values and FCs in treated- compared to non-treated samples. Wnt/β-catenin canonical pathway genes that are differentially expressed upon treatment with GSK3 inhibitor LY2090314 are rendered per IPA legend , as well as progenitor markers: p63α  and SOX9   A,B. On t < 0.01) C,D. The  < 0.01) D.p = 0.24), from treatment day 1 to day 3  and from day 4 to day 6 , whereas it was significant between day 7 and day 9 of treatment   A,B.Ex vivo expansion of cells from limbal biopsies is one of the most-used methods for CLET. The current setup is well suited for studying the effect of Wnt/β-catenin signaling on the self-renewal and/or differentiation status of the cells in ex vivo expanded hLESC cultures prior to transplantation. Previous studies have demonstrated that Wnt/β-catenin signaling activation maintains stemness and enhances proliferation in hLESC cultures isolated as single-cell cultures  ,30,31,33In embryonal stem cells, it was previously shown that Wnt/β-catenin signaling is repressed during self-renewal and that increased β-catenin induces differentiation identified by a higher expression of differentiation markers ,34. As eCEBPD gene is highly expressed in the human limbus compared to the cornea itself [The p63 marker has been for more than two decades the most-studied stemness/progenitor marker in hLESCs . Later, a itself . CEBP/δ a itself . Downrega itself . Similara itself . The absence of Cx43 and KRT3/12 differentiation markers is also used to indicate the stem cell nature of expanded hLESCs in vitro . Cx43 isAXIN2 gene and increased levels of total β-catenin in treated vs. untreated hLESC cultures. β-catenin has been reported to be upregulated during corneal wound healing [AXIN2 gene expression was found to be significantly upregulated in limbal tissue compared to the cornea [WNT2 and WNT16B remained low and unchanged upon LY2090314 treatment, whereas the levels of WNT6 and WNT11 were upregulated upon Wnt/β-catenin activation in hLESCs. These four Wnt ligands are predominantly expressed in the limbal area in situ [WNT11 has been reported to be more specific for the limbus than the cornea itself [WNT3 gene expression was upregulated in LY2090314 treated/Wnt/β-catenin-activated hLESCs in our study. Previously, WNT3 was reported to be significantly higher-expressed in the central cornea compared to the limbus [WNT7A downregulation upon Wnt/β-catenin activation during hLESC differentiation ex vivo. Interestingly, Wnt7A is found to maintain the corneal epithelium under controlled homeostasis, prevent skin-like epithelium formation, and determine hLESC differentiation to corneal epithelial cells through PAX6 in rabbits. [WNT1 upregulation in hLESCs upon the activation of Wnt/β-catenin signaling.The activation of Wnt/β-catenin signaling was confirmed by an upregulation of the  healing , wherease cornea . This su in situ ,56. WNT1a itself . WNT2 haa itself . WNT3 gee limbus . Inhibite limbus . Our resrabbits. . In linerabbits. . WNT5A wrabbits.  and diabrabbits. . We repoDKK1 was significantly downregulated in the treated samples, showing enhanced differentiation compared to controls. This correlates with previous findings where Dkk1 expression stimulated hLESC pluripotency and proliferation in cultures [DKK1 was predominantly expressed in the limbus compared to the cornea [cultures . DKK1 wae cornea ,63. Hence cornea . Howevere cornea . Similare cornea ,63, was e cornea .ALK7 is upregulated upon LY2090314 treatment of hLESCs following induced differentiation. Transforming growth factor β (TGFβ) seems to have an opposite role to Wnt/β-catenin signaling in hLESCs [Activin A receptor type 1C (ACVR1C), also known as ALK-7 (Activin receptor-like kinase 7), is a receptor binding to the ligands of the TGFβ family. ACVR1C phosphorylates directly to Smad-2/3 or Smad 1/5/8 and binds to Smad4 ligands, which then translocate to the nucleus and induce transcription . ALK-7 in hLESCs  and the n hLESCs . FZD7 is upregulated upon LY2090314 treatment in addition to increased proliferation and increased levels of β-catenin in treated hLESCs. The c-Myc oncogene maintains corneal epithelial architecture at homeostasis, modulates p63 expression, and enhances proliferation during tissue repair [Frizzled class receptor 1 (FZD7) is found exclusively expressed in the cells of the LBE ,63. FZD7e repair .Canonical and non-canonical Wnt ligands utilize similar systems to activate completely different cascade processes . By compActivation of the canonical Wnt/β-signaling in hLESCs was carried out through GSK-3 inhibition in our study, with the advantage of allowing the overview of spontaneous Wnt ligands and inhibitor down- and/or upregulation involved in hLESC differentiation. In contrast, previous studies have favored the Wnt/β-catenin signaling activation approach through particular Wnt ligand domination. We examined the effect of Wnt signaling activation in hLESC ex vivo cultures resulting in differentiation, reduced proliferation, and loss of stemness. This indicates the importance of regulating and avoiding activation of Wnt signaling to preserve stemness in hLESC cultures, findings similar to the dominance of the Wnt-signaling inhibitor DKK1 in the limbal stem cell niche in vivo . In futuIn conclusion, the present study confirms an increased differentiation and loss of stemness and proliferation upon persistent Wnt/β-catenin signaling activation with LY2090314, a GSK-3 inhibitor, in ex vivo expanded hLESCs from limbal biopsies. On the contrary, previous studies show increased stemness and proliferation upon Wnt/β-catenin signaling activation in hLESCs isolated as single-cell cultures. The comparative analysis of these two culture models evaluating Wnt/β-catenin signaling activation and inhibition would be very useful to elaborate on in future studies.


Among them, the Wnt signaling pathway is one of the most important pathways. It is not only involved in bone development and metabolism but also in the differentiation and proliferation of chondrocytes, mesenchymal stem cells, osteoclasts, and osteoblasts. Dkk-1 and SOST are Wnt inhibitory proteins that can inhibit the activation of the canonical Wnt signaling pathway and block the proliferation and differentiation of osteoblasts. Therefore, they may serve as potential targets for the treatment of osteoporosis. In this review, we analyzed the mechanisms of Wnt proteins, β-catenin, and signaling molecules in the process of signal transduction and summarized the relationship between the Wnt signaling pathway and bone-related cells. We hope to attract attention to the role of the Wnt signaling pathway in osteoporosis and offer new perspectives and approaches to making a diagnosis and giving treatment for osteoporosis. Osteoporosis, one of the serious health diseases, involves bone mass loss, bone density diminishing, and degeneration of bone microstructure, which is accompanied by a tendency toward bone fragility and a predisposition to fracture. More than 200 million people worldwide suffer from osteoporosis, and the cost of treating osteoporotic fractures is expected to reach at least $25 billion by 2025. The generation and development of osteoporosis are regulated by genetic factors and regulatory factors such as TGF- Osteoporosis is a serious, healthy disease that involves low bone mass and microstructure degeneration of bone tissue and is accompanied by a predisposition to fracture . FracturThe pathogenesis of osteoporosis is caused by osteoblast apoptosis and osteoclast proliferation in the body, which leads to bone metabolism disorder and bone loss, resulting in the deterioration of bone morphology . During β (TGF-β), parathyroid hormone (PTH), and fibroblast growth factor (FGF) [The formation and development of osteoporosis are regulated by various bone-related transcription factors and genetic factors at the molecular level, including transforming growth factor or (FGF) ,9,10. Thor (FGF) . When thβ is widely found in human normal tissues and cells, especially in bone tissues and platelets [β superfamily: TGF-β, activin, inhibin, bone morphogenetic protein (BMP), and so on. These family numbers are polypeptide compounds with similar structures and different functions. The newly secreted TGF-β gene (pre-pro-TGF-β) combines with the latency-associated protein (LAP) and forms a dimeric polypeptide chain on ribosomes, which has no biological activity, that is, the inactive precursor protein (pro-TGF-β). After entering the Golgi apparatus, pro-TGF-β is further processed, modified, and secreted from cells. Finally, the pro-TGF-β is hydrolyzed by a protease or non-protease to form mature TGF-β with biological activity. Generally, pro-TGF-β releases bioactive TGF-β under acidic conditions or after being cleaved by matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9). Then, it recruits mesenchymal stem cells (MSCs) to the site of bone reconstruction to promote the differentiation and proliferation of osteoblasts and new bone formation. The recruitment of MSCs by TGF-β is mainly achieved through the ALK5-Smad2/3-Smad4 signaling pathway. Smad3 plays a major role in this process, while Smad2 plays a minor role [TGF-latelets . There anor role .β molecules in mammals, with similar structures but different genetic codes: TGF-β1, TGF-β2, and TGF-β3. The potential TGF-β1 is secreted in almost all cells. TGF-β2 and TGF-β3 are mainly found in epithelial cells and mesenchymal cells, respectively. TGF-β1 can not only promote the proliferation of osteoblasts but also induce the differentiation and maturation of osteoblasts [β2 binding with TPRI and TPRII on BMSCs’ membrane forms a complex of TGF-β2/TPRI/TPRII to activate TPRI and then accelerate the formation of osteoblasts through the Smad protein [β3 can interact with the receptors of TGF-β3 on the osteoblast membrane and form phosphorylated functional complexes, which promote the phosphorylated Smad2 and Smad3 proteins to enter the nucleus and regulate the synthesis of transcription factors Runx2 to affect the cellular transcription [There are three main subtypes of TGF-eoblasts . It can  protein . TGF-β3 cription .β usually exists as a dimer linked by disulfide bonds. Compared with other growth factors, TGF-β mildly regulates cell proliferation and differentiation and depends on the dose of TGF-β, cell cycle, and cell type. Recent studies showed that low concentrations of TGF-β enhanced the proliferation and differentiation of osteoblasts, while high concentrations of TGF-β suppressed the proliferation and differentiation of osteoblasts [β to maximize its side effects needs to be further studied. The effect of TGF-β on osteoblasts is time-dependent and plays different roles at different stages of cell differentiation. TGF-β can promote the proliferation, early differentiation, and matrix production of the osteoblast progenitor cells. However, in the later stage, Smad3 inhibits the expression and function of Runx2, and thereby TGF-β inhibits the differentiation of osteoblasts and matrix synthesis [TGF-eoblasts ,18,19. Hynthesis .β is mainly synthesized by osteoblasts, osteoclasts, and chondrocytes through autocrine and paracrine pathways [β plays an important role in regulating the function of osteoblasts [β can also induce the differentiation of MSCs into osteoblasts by activating extracellular signal-related kinases, such as c-Jun N-terminal kinase and mitogen-activated protein kinase-P38 [β can inhibit the differentiation and formation of osteoclasts, thereby inhibiting bone resorption [β, stimulate osteoblasts to down-regulate the RANKL expression, and increase the OPG expression to inhibit the osteoclast differentiation of BMSCs [β1 mRNA and ALP activity increased obviously after the addition of an osteogenic inducer to MSCs, which significantly promoted bone formation [In bone tissue, TGF-pathways . It can eoblasts . It not nase-P38 ,24. In asorption . Quinn eof BMSCs ,27. An iormation ,29.β participates in the whole process of cartilage occurrence and development [β promotes chondrocyte differentiation and inhibits chondrocyte hypertrophy differentiation to ensure the amount of extracellular matrix in articular cartilage. In the late stage of chondrogenesis, the expression of TGF-β decreases, and the synthesis of some degradation enzyme inhibitors is induced to maintain the chondrocyte phenotype to ensure the stability and function of articular cartilage, which plays an important role in maintaining the integrity of articular cartilage. In addition, TGF-β can effectively regulate the processes of bone resorption and bone remodeling and also has a great regulatory effect on osteoblast replication and bone matrix synthesis [β is an important factor in the process of bone formation and absorption, which can stimulate the synthesis of collagen by osteoblasts and osteoblasts, increase the synthesis of cartilage-specific type I collagen and fibronectin, and reduce the synthesis of type II and type X collagen. It can also promote the proliferation and differentiation of mesenchymal cells and osteoblasts. By regulating the expression of proteins, TGF-β promotes the secretion of fibronectin and collagen by fibroblasts and accelerates the formation of bone and cartilage.TGF-elopment . In the ynthesis ,32. TGF-β superfamily; more than 30 family members have been found; the molecular weight is about 30 kDa [β family. It not only lacks the conserved region structure characteristic of the TGF-β family but is also a zinc-dependent metalloproteinase, thus it is a completely different protein [Urist found out in 1965 that the bone matrix contained active proteins that promoted the formation of cartilage and bone tissue . This act 30 kDa . They ar protein . The BMP protein ,39. Stud protein .β-catenin, accelerate the phosphorylation of β-catenin, and increase the concentration of β-catenin [β-catenin pathway. Mechanism studies showed that the BMP-2/Smad signaling pathway may affect the activity of the β-catenin intracellular regulatory complex GSK-3β-APC-Axin through Smurf2. This prevents the degradation of β-catenin, thereby enhancing the activity of the Wnt/β-catenin pathway [Wnt and BMP have their ligands, receptors, transduction proteins, and molecular mechanisms, but they interact and influence each other in the process of regulating cell differentiation and proliferation ,42. Ther-catenin . It is i pathway .β-catenin played an important role. In vivo experiments confirmed that BMP-9-induced ectopic osteogenesis and calcium salt deposition could be inhibited by FRZ overexpression or β-catenin knockdown.Wnt and BMP proteins, two important signaling molecules, participate in the regulation of bone regeneration, the promotion of stem cell proliferation, and osteogenic differentiation through synergistic effects ,47. Li eβ-catenin signaling partially stimulates chondrocyte differentiation through the BMP-2/4 signaling pathway and BMP-2 promotes/inhibits the expression of Runx2 in a dose-dependent manner, thereby affecting protein ubiquitination in chondrocytes.BMP-2, 4, 5, 6, 7, and 14 are proteins closely related to chondrogenesis and development ,51. BMP-β-catenin and BMP-9 signaling pathways are involved in the osteogenic differentiation of apical dental papilla stem cells [β-catenin can down-regulate the effect of BMP-9 on early and late ISCAP osteogenesis. Although BMP-9 has strong potential to become a new method to enhance bone reconstruction in the clinic, its mechanism has not yet been fully understood. Therefore, further research is needed to ensure that BMP-9 can be safely and effectively applied in clinics.Both Wnt and BMP signaling pathways participate in tooth development . They muem cells . BMP-9 cBMP is important for bone growth because it is an acidic glycoprotein with strong osteogenic properties. This growth factor with multifunctional differentiation participates in the processes of bone formation, differentiation, and absorption and has different degrees of influence on the differentiation and proliferation of osteoblasts, osteoclasts, and chondrocytes . It playThe FGF family consists of twenty-three members with molecular weights ranging from 17 kDa to 34 kDa (in vertebrates) . They arThe fibroblast growth factor (FGF) is widely found in the human bone matrix. As a potential mitogen, it can accelerate cartilage repair in vivo and promote the proliferation, differentiation, and maturation of chondrocytes in vitro ,69. The FGF-2 expression in chondrocytes and osteoblasts affects the maturation and development of primary cartilage and growth plate cartilage and cartilage regeneration and significantly stimulates the proliferation and osteogenic ability of BMSCs at the early stage of differentiation ,73,74. TFGF-23 is a new member of the FGF family with a molecular weight of 32 kDa. It is mainly secreted by bone cells and regulates calcium and phosphorus metabolism by acting on the kidney . FGF-23 FGF-11 is a factor driving pathological bone resorption in osteolytic diseases, which can be induced by FGF-11 to stimulate bone resorption under hypoxic conditions . It is iThe human FGFR family includes four members: FGFR-1, FGFR-2, FGFR-3, and FGFR-4 . AlthougFGFR is an important signal molecule regulating the development of bone gaps. Its mutation will lead to the development of bone gaps, including achondroplasia, premature closure syndrome of the nape suture, and abnormal metabolism syndrome ,100,101.β-catenin pathway, the non-Wnt/Ca2+ pathway, and the Wnt planar cell polarity (PCP) pathway [Until now, three Wnt signaling pathways have been found, including the Wnt/ pathway . An incr pathway ,109. It  pathway . Recent  pathway .(1)Wnt proteinsThe Wnt gene was discovered by Nusse when studying mouse breast cancer and was named lnt-1 at that time . FurtherTo date, more than nineteen Wnt proteins have been discovered  , which aβ-cateninn pathway [β-catenin pathway [β-catenin signaling blocks the entry of β-catenin into the nucleus to activate TCF-1, thus failing to activate Runx2. Therefore, when stem cells from the apical papilla (SCAP) are infected with Wnt3a and BMP-9, the osteogenic ability of SCAP cells is significantly enhanced. However, the synergistic mechanism between Wnt3a and BMP-9 needs to be further studied. At the late stage of osteoblast development, the expression of Wnt3b and β-catenin is increased, the activity of RANKL is inhibited, and both the number and activity of osteoclasts are decreased.Wnt signaling is a key molecule that determines the directional differentiation of MSCs into osteoblasts and participates in bone formation, development, and remodeling ,119. It  pathway ,122,123. pathway . The mec pathway . Silenciβ-catenin pathway but also the Wnt/Ca2+ pathway. Which pathway is activated depends on the receptor type on the cell surface. When Wnt5a binds to the Ror2 receptor, it inhibits β-catenin transcriptional activity and subsequently blocks the canonical pathway [β-cetenin, suppress 3T-L1 differentiation, and induce osteogenic differentiation [Wnt5a is essential for maintaining the osteogenic differentiation of human MSCs and acts through mediating Lim-only protein FHL2 ,127,128. pathway . When Wn pathway . In addintiation . (2)β-cateninβ-catenin is located on human chromosome 3p22-p21.3 and was first discovered by Walt, a German biologist. It is a multifunctional protein with a molecular weight of 88 kDa and is composed of 781 amino acids. It contains one carbon-terminal domain, one nitrogen-terminal domain, and twelve armadillo (ARM) repeats. The carboxy-terminal domain consists of 100 amino acids and contains a catenin binding site as well as GSK-3β and CKI phosphorylation sites, which regulate the transcription of downstream target genes. ARM repeats are conserved regions in β-catenin. They are composed of 36 α-helices enclosed by rod-like superhelices, which can bind to E-cadherin, Axin, and APC proteins. These proteins can not only improve the stability of the β-catenin structure but also coordinate the interaction between β-catenin and GSK-3β [β-catenin. β-catenin is the core molecule of the canonical Wnt signaling pathway, which is responsible for transmitting upstream and downstream molecular signaling.d GSK-3β . The amiβ-catenin is mainly distributed in the cell membrane or in the form of a complex in the cytoplasm. A small amount of free β-catenin molecules can be easily degraded by proteases in the cytoplasm [β-catenin and E-cadherin have been shown to participate in signal transduction between different cells. In the absence of the Wnt ligand, β-catenin mostly binds to E-cadherin in the cell membrane and makes it attach to the cytoskeletal protein actin, which mediates cell-cell adhesion. When the expression of β-catenin was up-regulated, the adhesion between cells was enhanced, and the invasion and metastasis of cells were not easy to occur in the cancer environment. On the contrary, when the expression of β-catenin is down-regulated, the adhesion between cells is destroyed, and the cells are more susceptible to invasion and metastasis. For a small amount of β-catenin, Axin in the APC/Axin/GSK-3 complex forces GSK-3β to move closer to β-catenin and together with APC promotes the phosphorylation of β-catenin, which is finally degraded through the ubiquitin-proteasome pathway. At this time, TCF/LEF binds with different transcriptional repressor proteins and inhibits the expression of Wnt target genes [β-catenin is related to cell type and organ type. Particularly, in cancer cells, the change in β-catenin concentration is more obvious.As an adhesive molecule, ytoplasm . The proet genes . The conβ-catenin has a direct effect on the osteoblastic precursor cells and osteoblasts [β-catenin may indirectly induce MSCs to differentiate into osteoblasts by enhancing their response to BMP-2 [β-catenin stimulates the activation of the OPG gene promoter in osteoblasts, thereby promoting osteoblast differentiation [β-catenin is related to the regulation of cartilage formation and differentiation not only in the early embryonic stage but also in postnatal cartilage and bone formation. The concentration of β-catenin mRNA and protein increases obviously in OA patients and mechanically injured human articular cartilage tissues [β-catenin in articular chondrocytes show decreased articular cartilage tissue, cleft formation, fibrosis, and osteophytes [β-catenin is knocked down, cartilage enlargement and maturation are inhibited [β-catenin may act by activating the Ihh and Runx2 signaling or inhibiting PTHrP and IGF-1 signaling [Generally speaking, eoblasts . As an ito BMP-2 . Howeverntiation . In addi tissues . Transgeeophytes . When β-nhibited . The mecignaling ,144.(3)β signal transductionMechanisms of Wnt/β-catenin pathway, β-catenin is the core component. As shown in β-catenin in the cytoplasm binds to E-cadherin in the cell membrane and makes it attach to the cytoskeletal protein actin to mediate cell-cell adhesion. A small amount of β-catenin forms destruction complexes with Axin, APC, CKI, and GSK-3β. Axin in the complex makes GSK-3β approach β-catenin, and together with APC, accelerates the phosphorylation of β-catenin, then combines with beta-transduction repeats-containing E3 ubiquitin ligase protein (β-TRCP), and finally is degraded by the ubiquitin-proteasome pathway, so that β-catenin in the cytoplasm remains at a low level. Meanwhile, TCF/LEF binds to a variety of transcriptional repressor proteins, such as Groucho family members, to prevent the expression of Wnt target genes.In the Wnt/Dickkopf1 (Dkk-1), secreted frizzled-related protein (SFRP), sclerostin (SOST), and Wnt inhibitor factor 1 (Wif1) are all secreted Wnt inhibitor proteins that can inhibit the activation of the canonical Wnt signaling pathway . Dkk-1 aβ-catenin signaling pathway is activated  in the nucleus and convene the transcription activator, thus initiating the transcription of the target gene downstream of the Wnt pathway and expressing cell cycle proteins, such as cyclin D1, Runx2, Osx and MMP. The Wnt signaling pathway regulates gene transcription through the combination of β-catenin and TCF/LET family, the key factor of which is to accumulate enough β-catenin in the cytoplasm. When the β-catenin level in the cytoplasm is low, the Wnt signaling pathway is turned off. When the level is high, the Wnt signaling pathway is turned on.In the presence of a Wnt molecule, the Wnt/ctivated . The Wnt2+ pathway are Wnt ligand, FRZ receptor, G-protein, DVL protein, PLC, IP3, DAG, PKC, CaM, CaMK Ⅱ, and other components. It participates in regulating the concentration of intracellular Ca2+. In the presence of Wnt signaling, the Wnt ligand binds FRZ receptors on the cell membrane to recruit DSH. Subsequently, the trimeric G-protein Gα/Gβ/Gγ binding with DSH activates PLC, which acts on phosphatidylinositol-4,5-diphosphate in the cell membrane to produce inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 activates Ca2+ after entering the endoplasmic reticulum, which can promote the increase of intracellular Ca2+ concentration, accelerate the formation of the Ca2+/CaM complex, and thus activate downstream calcineurin (CaN). Activated CaN dephosphorylates NFAT in cells, then migrates to the nucleus to act on downstream target molecules. DAG activates protein kinase C (PKC) and promotes CaMKⅡ to exert biological effects through autophosphorylation. In general, the Wnt/Ca2+ pathway, based on the increase of intracellular Ca2+ concentration, activates PKC, CaMK Ⅱ, NFAT, and other signaling and transcription factors and plays a role in regulating cell adhesion and gene expression [Proteins involved in the Wnt/Capression . 2+ signaling pathway is mainly activated by Wnt5a and Wnt11 [2+ temporarily increase, which triggers the activation of NF-κB and NFAT to regulate the generation of osteoclasts. The Wnt5a/Frizzled-2/Ca2+ pathway has an inhibitory effect on the Wnt/β-catenin signaling pathway. In the downstream of the Wnt5a/Frizzled-2/Ca2+ pathway, PKC can activate GTPase Cdc42, while CAMK Ⅱ can phosphorylate TGF-β-activated kinase 1 (TAK1). It induces the activation of Nemo-like kinase (NLK) and inhibits the transcription of the Wnt/β-catenin signaling pathway. Wnt11 participates in the differentiation of MSCs. The stable expression of Wnt11 can increase the concentration of intracellular free calcium ions, thereby promoting the activity of PKC and inhibiting NF-κB activity. Wang et al. found that SPA (S.aureus protein A) could inhibit the non-canonical Wnt/Ca2+ signaling pathway mediated by Wnt11 and significantly downregulate the ability of hMSCs to differentiate into osteoblasts in vitro [The Wnt/Cand Wnt11 . Wnt5a ind Wnt11 . When Wnin vitro . Wnt11 pThe Wnt/PCP pathway is a dishevelled (DSH)-dependent pathway mediated by c-jun N-terminal kinase (JNK). It has two signal transduction pathways. The first pathway is that the Wnt protein binds with the FRZ receptor, the Ror2 receptor, or the Ryk receptor to promote DSH to recruit disheveled-associated activator of morphogenesis 1 (DAMM1) and mediates Rho to active Rho-associated protein kinase (Rock), thereby causing cell migration, cytoskeleton, and tissue structure changes. Another pathway is that DSH directly cooperates with Rac1 and JNK to activate nuclear factor c-Jun and activate transcription factor 2 (ATF2), causing transcription, translation, and expression of downstream target genes, thereby regulating cell differentiation and proliferation .The Wnt/PCP pathway is mainly activated by Wnt4, Wnt5a, and Wnt11, of which Wnt5a is most closely related to bone formation. Wnt5a regulates the change of the cytoskeleton by regulating the activity of Rock , thus inβ-catenin signaling pathway. Overexpression of Wnt10a increases the relative expression of β-catenin and then induces osteogenic differentiation. The Wnt signaling pathway is closely related to BMSCs, osteoblasts, osteoclasts, and chondrocytes . BMSCs aβ-catenin pathway. Hang et al. studied the effect of the Apelin protein on the osteogenesis of hBMSCs. The results showed that the expression of β-catenin was up-regulated and the mineral deposition increased in the Apelin-treated cells. Inhibitors that specifically inhibit the Wnt/β-catenin signaling pathway also decreased Apelin-induced osteogenic differentiation. Therefore, Apelin partially regulates the osteogenic differentiation of BMSCs and effectively promotes fracture healing through the Wnt/β-catenin signaling pathway [The osteogenic differentiation of BMSCs is regulated by biological molecules such as proteins, RNAs, and cytokines through the Wnt/ pathway . β-catenin signaling pathway [β-catenin expression and TCF/LEF transcriptional activity in osteoblasts [β-catenin, and LEF-1, and accelerate fracture healing, which indicates that the Wnt/β-catenin signaling pathway plays positive roles in regulating osteogenic differentiation in vivo and in vitro [The canonical Wnt signaling pathway plays a crucial role in the differentiation, proliferation, and maturation of osteoblasts and is deeply involved in regulating bone development and bone remodeling. Kim et al. found that albiflorin plays a positive role in osteoblast differentiation in MC3T3-E1 cells. It enhances the expression of bone formation-related genes such as OSN, OCN, and OPN through the Wnt/ pathway . During  pathway . Intraveeoblasts . Guhong in vitro . β-catenin is closely related to osteoblast activity in type I diabetic animals. Moderate activation of β-catenin signaling in osteoblasts can significantly enhance bone formation ability in type 1 diabetic mice and effectively improve bone loss and bone strength damage [β-catenin gene can block the translocation of β-catenin into the nucleus and activate TCF-1, thereby failing to activate Ru. Therefore, when the ISCAPs are infected with Wnt3a or BMP-9 alone or in combination, the osteogenic differentiation ability of ISCAPs is significantly stronger than that of ISCAP cells with β-catenin silencing [The expression of h damage . In addiilencing . Wnt sigilencing . The embilencing .β-catenin pathway. The concentration of SOST in osteoblasts and osteocytes is negatively related to bone mass and bone strength. In animal models of osteoporosis, inhibition of SOST by the monoclonal antibodies omosozumab and blosozumab induced osteoblast differentiation and new bone formation, normalized bone mass, and improved bone structure and strength. Clinical trials have also shown that anti-sclerostin antibody treatment can significantly increase bone formation and reduce bone resorption [β-catenin signaling and significantly improve bone mass. Therefore, PTH may serve as a potential drug for the treatment of postmenopausal osteoporosis [β-catenin levels, inhibits osteoblast differentiation, and promotes osteoblast apoptosis. Dkk-1 binds to the Wnt receptor LRP5/6 on the surface of osteoblasts, blocking the Wnt signaling pathway and preventing osteoblast proliferation and differentiation. Qiang et al. found that the number of osteoblasts decreased by 49% and the concentration of serum osteocalcin decreased by 45% in transgenic mice with Dkk-1 overexpression, resulting in serious bone loss [The Wnt signaling pathway enables mesenchymal stem cells to conduct downward along the osteoblast differentiation pathway. Sclerostin (SOST) is an important signaling molecule produced in the Wnt/sorption . Parathyoporosis . In osteoporosis . Overexpone loss . On the one loss . The relevant evidence of Wnt regulation of osteoclasts is mainly indirect , meaningβ-catenin significantly increases the number of osteoclasts, enhances bone resorption, and sharply reduces bone mass, which causes osteoporosis. On the contrary, overexpression of β-catenin causes a significant increase in the number of osteoblasts and bone mass and enhances bone formation. Therefore, the activation of the Wnt/β-catenin signaling pathway can provide a new approach to treating osteoporosis [Osteoporosis is a common bone disease that is mainly manifested as decreased bone mass, bone mineral density, bone strength, and increased risk of fracture. Wnt plays an irreplaceable role in bone development, osteoblast differentiation and maturation, and maintenance of normal bone homeostasis. The activation of Wnt signaling can lead to dual effects on the treatment of bone diseases . Knockdooporosis . β-helix domain of LRP5/6, thereby regulating the transcription of downstream target genes. Heiland found that inhibition of Dkk-1 had an inhibitory effect on inflammatory bone loss in TNF transgenic mice, accompanied by an increase in the expression level of β-catenin [β-catenin, accompanied by the decreased bone formation and increased bone resorption [The bone resorption inhibitors include bisphosphonates, estrogen calcitonin, selective estrogen receptor modulators, and Wnt inhibitors. Dickkopf-1 (Dkk-1), an important inhibitor of the Wnt signaling pathway, is involved in bone development and bone remodeling. It inhibits Wnt signaling by competitively binding to the -catenin . This me-catenin . In addisorption . These sβ-catenin signaling could effectively improve osteoporosis caused by thalassemia. At present, the research on the Dkk-1 antibody is mainly limited to animal experiments. How to specifically regulate the expression of Dkk-l in bone tissue without affecting the metabolism of other tissues and organs is an urgent problem to be solved.Anti-Dkk-1 antibodies can effectively alleviate osteoporosis symptoms caused by estrogen deficiency. Glantschnig et al. studied the therapeutic effect of monoclonal anti-Dkk-1 antibodies (RH2-18) on osteoporosis mice caused by estrogen deficiency . After 8The dynamic balance of bone metabolism plays a significant role in maintaining the function of normal bone tissue. When the balance is broken, bone metabolic diseases such as osteoporosis will occur. The Wnt signaling pathway participates in bone development and metabolism and is closely associated with the differentiation and proliferation of MSCs, osteoblasts, osteoclasts, and chondrocytes. Based on the role of the Wnt signaling pathway in bone metabolism, an increasing number of researchers pay attention to the pathogenesis of osteoporosis and develop drugs for the treatment of osteoporosis by inhibiting or activating the Wnt signaling pathway. SOST and Dkk-1 can competitively bind to the co-receptor LRP5/6 in the Wnt signaling pathway and then regulate the transcription of downstream target genes, ultimately leading to the occurrence and development of osteoporosis. Therefore, SOST and Dkk-1 have become very potent targets for the treatment of osteoporosis. Inhibiting the expression of SOST and Dkk-1 and activating the Wnt signaling pathway can effectively increase bone formation and inhibit bone resorption, which is considered an effective method to treat osteoporosis. Compared with traditional anti-resorption drugs, anti-SOST antibodies, and anti-Dkk-1 antibodies can effectively increase bone formation and reverse the decline of bone mineral density in osteoporosis patients. However, the specific mechanism is still unclear due to the complexity of the Wnt signaling pathway. The clinical use of antagonists that inhibit signaling pathways is limited. In addition, the process of bone metabolism is not only regulated by the Wnt pathway but also involves the Notch pathway, Hedgehog pathway, and OPG/RANKL/RANK pathway, and each signaling pathway also affects the other. Therefore, in-depth studies of the mechanism of the Wnt pathway in bone metabolism and its relationship with other signaling pathways will provide new methods to prevent and treat osteoporosis.


The receptor for activated C kinase 1 (RACK1) is a key scaffolding protein with multifunctional and multifaceted properties. By mediating protein-protein interactions, RACK1 integrates multiple intracellular signals involved in the regulation of various physiological and pathological processes. Dysregulation of RACK1 has been implicated in the initiation and progression of many tumors. However, the exact function of RACK1 in cancer cellular processes, especially in proliferation, remains controversial. Here, we show that RACK1 is required for breast cancer cell proliferation in vitro and tumor growth in vivo. This effect of RACK1 is associated with its ability to enhance β-catenin stability and activate the canonical WNT signaling pathway in breast cancer cells. We identified PSMD2, a key component of the proteasome, as a novel binding partner for RACK1 and β-catenin. Interestingly, although there is no interaction between RACK1 and β-catenin, RACK1 binds PSMD2 competitively with β-catenin. Moreover, RACK1 prevents ubiquitinated β-catenin from binding to PSMD2, thereby protecting β-catenin from proteasomal degradation. Collectively, our findings uncover a novel mechanism by which RACK1 increases β-catenin stability and promotes breast cancer proliferation. The receptor for activated C kinase 1 (RACK1) is a member of the tryptophan-aspartate repeat (WDR) protein family . SubsequDysregulation of RACK1 has been implicated in the initiation and progression of various cancers. In most cases, RACK1 has been reported to promote proliferation, radiation and chemotherapy resistance, invasion and metastasis in a variety of cancers, such as lung, liver, breast, esophageal, melanoma, glioma, myeloma and neuroblastoma –29. ConsThe mechanism whereby RACK1 regulates cancer progression remains to be elucidated. As a signaling hub, RACK1 has been associated with the activation of several cancer-related cellular signaling pathways, including PKC , receptoOur previous studies have shown that RACK1 is essential for the drug resistance and metastasis of breast cancer cells . In thishttps://score.depmap.sanger.ac.uk/), RACK1 affected the viability of 98% of cell lines, including 41 breast cancer cell lines  assay using anti-Flag antibodies in previously obtained RACK1-flag-expressing MDA-231 cells and analyzed the IP product by mass spectrometry Fig. . A totalNext, we determined whether RACK1 silencing affects β-catenin mRNA expression in two breast cancer cell lines. As shown in Fig. β-catenin is known to be phosphorylated by GSK3β prior to ubiquitin-mediated proteasomal degradation . A previNext, we further analyzed the RACK1 interactome and found that several proteasome components were highly enriched in these interacting proteins, with PSMD2 having the highest content and score Fig. . A Co-IPSince PSMD2 is a key subunit of the 19S proteasome, it acts as a ubiquitin receptor that recognizes substrates and delivers them to the 20S core particle for degradation . While βTo investigate the underlying mechanism by which PSMD2 regulates β-catenin stability, as shown in Fig. To identify the specific domain of the PSMD2 protein involved in the interaction with RACK1 and β-catenin, we performed molecular docking analysis and found that the Armadillo-like helical domain (355 to 853 amino acids) may play a key role. Molecular docking revealed that several sites of PSMD2  might be involved in the interaction with RACK1 Fig. , and othThe present study supports the tumor-promoting role of RACK1 in breast cancer. Our in vitro and in vivo evidence suggests that RACK1 is required for breast cancer cell proliferation by regulating cell cycle progression. We uncovered that RACK1 promotes the stability of β-catenin, thereby facilitating the activation of the canonical WNT pathway in breast cancer cells. We identified PSMD2 as a novel binding partner for RACK1 and β-catenin. Mechanistic investigation demonstrated that RACK1 interacts with PSMD2 and prevents ubiquitinated β-catenin from binding to PSMD2, thereby protecting β-catenin from UPS-mediated degradation. Collectively, our findings suggest that RACK1 facilitates breast cancer progression by competitively inhibiting β-catenin binding to PSMD2, increasing β-catenin stability, and promoting WNT pathway activation Fig. .Fig. 8DiAlthough RACK1 has been implicated in the initiation and progression of many tumors, the exact function of RACK1 in cancer cellular processes, particularly in proliferation, remains controversial. Initial studies showed that elevated RACK1 inhibited the proliferation of transformed NIH-3T3 cells . Subsequβ-catenin is a well-known key proliferation-related regulator downstream of the WNT pathway, and its stability is regulated by the UPS . Here, wThe mechanisms by which RACK1 regulates β-catenin stability in breast cancer cells are worthy of investigation. Although a previous study has shown that in gastric cancer cells, RACK1 promotes β-catenin degradation by interacting with GSK3β and stabilizing the β-catenin destruction complex . NeverthIn conclusion, our results suggest that RACK1 facilitates breast cancer progression by enhancing the stability of β-catenin. We identified PSMD2 as a novel binding partner of RACK1 and β-catenin, and although RACK1 did not bind to β-catenin, it inhibited the interaction of PSMD2 with β-catenin and the subsequent proteasome-mediated degradation. Thus, we reveal a novel mode of interaction between PSMD2, RACK1 and β-catenin, that may be a mechanism involved in regulating the activation of the WNT pathway, and targeting this pathway may be an effective way to treat breast cancer.2 at 37 °C).Human breast cancer cell lines MDA-MB-231 (MDA-231), BT549 and human embryonic kidney 293T (HEK-293T) were purchased from the American Type Culture Collection . MDA-231 and BT549 cells were grown in RPMI-1640 medium ; HEK-293T cells were cultured in DMEM medium, and all media were supplemented with 10% fetal bovine serum (HyClone) under standard culture conditions  and the other targets its non-coding region (shRACK1-#2). The β-catenin-specific shRNA sequences were cloned into the lentiviral vector pLKO.1- puromycin of the BamH I and Age I cloning sites. The shRNA sequences are shown in Supplementary Table Isolation, quantification, SDS-PAGE and transfer protocols for total cellular proteins were performed as previously described . BrieflyThe detailed method for quantitative real-time PCR (qRT-PCR) was performed as described previously . BrieflyCell proliferation capacity was determined by the Cell Count Kit 8 (CCK8) assay and the colony formation assay. The CCK8 assay was carried out as previously described . Briefly6 cells were injected orthotopically into the mammary fat pad. Seven days after the inoculation, the tumors became detectable. Tumor volume was measured every 3 days and the mice were sacrificed after 35 days. Tumor volume (cm3) was measured by using the formula: 0.5 × smallest diameter × largest diameter2. The tumors were then dissected, cleaned, imaged, fixed in formalin, embedded in paraffin, and serially sectioned. The expression of RACK1, β-catenin and Ki67 was detected by immunohistochemical staining according to a standard protocol as described previously. The following antibodies were used in this experiment. RACK1 , β-catenin , and Ki67 . All experimental operations complied with the requirements of the Animal Ethics Committee of Tianjin Medical University Cancer Institute and Hospital.Four- to five-week-old of female BALB/nude mice were purchased from Gempharmatech Co., Ltd  and randomized into four groups. Approximately 5 × 10Cells were fixed with 75% ethanol at 4 °C overnight. Cells were then centrifuged, washed with PBS, and stained with propidium iodide (50 μg/mL) in the presence of RNase A for 30 min at 37 °C. A total of 30,000 cells were analyzed using a FACScan flow cytometer in triplicate .3VO4, 2 mM EDTA, and 1 × protease inhibitor cocktail), and the lysates were centrifuged at 12,000 × g for 15 min at 4 °C. The supernatant was precleared with protein A/G agarose (Millipore) and then incubated with 1 μg of antibody overnight at 4 °C. The immunocomplex was incubated with protein A/G agarose beads for 1 h at room temperature. The beads were then washed three times with cell lysis buffer. The final pellets were resuspended in 1× SDS sample buffer, and then analyzed by Western blotting.Co-IP analysis was performed as described previously . In brie4HCO3, dehydrated with 100% acetonitrile (ACN), and then reduced with 10 mM dithiothreitol. Afterwards, the samples were dehydrated again in 100% ACN and alkylated with 55 mM iodoacetamide. After washing with 50 mM NH4HCO3, the samples were dehydrated with ACN, and digested with trypsin overnight at 37 °C. Tryptic peptides were extracted with 50% ACN/5% formic acid (FA), dried to the completeness and resuspended in 2% ACN/0.1% FA. Then, the peptides were dissolved in 0.1% FA (solvent A), loaded directly onto a home-made reversed-phase analytical column , and eluted on an EASY-nLC 1000 UPLC system at a constant flow rate of 400 nL/min. The gradient consisted of increasing from 6% to 23% solvent B  in 16 minutes, increasing from 23% to 35% in 8 minutes, climbing to 80% in 3 minutes, and then remaining at 80% for the final 3 minutes. The eluted peptides were then subjected to the NSI source and analyzed by tandem mass spectrometry (MS/MS) on a Q ExactiveTM Plus (Thermo) coupled online to the UPLC. Finally, the resulting MS/MS data were processed using Proteome Discoverer 1.3. The tandem mass spectra were searched against the KEGG database. Raw data has been provided in the Supplemental Table Tandem mass-tag (TMT)-based quantitative proteomics analysis was performed by PTM Biolabs  as described previously . In brieThe dual-luciferase reporter assay was performed using a commercial kit  following the manufacturer’s instructions. In brief, cells were plated in triplicate in 6-well plates. The next day, 2.5 ng of firefly luciferase vector (TOPflash or FOPflash) and 0.25 ng of Renilla luciferase vector were co-transfected into each well of cells, and 48 hours later, cell lysates were collected and measured by luminometer, repeated three times. Raw data has been provided in the Supplemental Table https://www.rcsb.org/). All protein structures were processed in the Molecular Operating Environment (MOE 2019.1) platform with Amber10 stand selection, including removal of water and ions, protonation, addition of missing atoms and complementation of missing groups, and protein energy minimization. Using HDOCK software, the protein was set to rigid, the docking contact site was set to full surface, the conformation generated after docking was set to 100, and the conformation with the most negative energy was selected using the scoring function. Pymol 2.1 software was used for visualization.The 3D structures of RACK1 (PDB ID: 4AOW), β-catenin (PDB ID: 7AR4), and PSMD2 (PDB ID: 6MSD) were obtained from the RCSB database  was used to determine the protein expression of RACK1 and RACK1 co-expression genes. The statistical software R (version 3.4.1) and the R packages “ggplot2” and “corrplot” were used for statistical analysis and visualization of the data [P < 0.05 was considered to indicate a statistically significant difference.The publicly available TCPA-BRCA database (the data . Other saj-checklistEthics approval and consentsupplemental figure1supplemental figure2supplemental figure3supplemental figure4supplementary figure legendsSupplementary Table1Supplementary Table2Supplementary Table3